Receptors for insulin-like growth factor-I (IGF-I) in myocardial capillary endothelium of the intact perfused heart

Isolated intact, beating hearts were perfused with HPLC-pure [125]-IGF-I (1 ng/ml) alone or [125]-IGF-I (1 ng/ml) plus varying concentrations of unlabeled IGF-I (10-3,000 ng/ml) or unlabeled insulin (1,000-100,000 ng/ml). After 1 min of perfusion with peptides, the hearts were rapidly fixed, sectioned and analyzed for radioautographic [125I] grain counts. Greater than 90% of [125I] grains were shown to represent intact [125I]-IGF. Maximal grain counts over capillaries occurred after perfusion with [125I]-IGF-I alone and decreased in a dose-dependent manner when unlabeled IGF-I was coperfused. Coperfusion of [125I]-IGF-I with unlabeled insulin also decreased 125I grains over capillaries but less potently than unlabeled IGF-I. EM radioautography demonstrated that [125I]-IGF-I grains were localized over capillary endothelial cells. Thus, specific IGF-I receptors are present in the capillary endothelium of the intact heart and have properties similar to IGF-I receptors in cultured capillary endothelial cells.     

Insulin-like growth factor I (IGF-I) and insulin binding to erythrocytes of normal prepubertal children and adults.

Erythrocyte insulin-like growth factor I (IGF-I) and insulin receptors were characterized in 10 normal prepubertal children (5 girls and 5 boys) aged 4-11 yrs and 10 normal adults (4 women and 6 men) aged 32-47 yrs. erythrocytes were purified from 5 ml of blood by Ficoll-Paque gradient centrifugation. Reticulocytes count in the erythrocyte suspensions were lower than 1%. Insulin and IGF-I binding assays were performed simultaneously. Maximal percent binding of [125I] labelled IGF-I was significantly higher in prepubertal children than in adults (8.7 +/- 0.7% versus 6.2 +/- 0.5% at a concentration of 5 x 10(9) erythrocytes/ml). Scatchard analysis revealed the high affinity constant was better in prepubertal children (Ka = 4.6 +/- 1.3 nM-1 versus 1.8 +/- 0.2 nM-1), whereas the binding capacity was similar (5.8 +/- 1.1 versus 7.7 +/- 0.8 high affinity binding sites/cell). In both groups, unlabelled IGF-I inhibited tracer-binding half maximally at about 1 nM. Insulin was 100-fold less potent. In adults, specific binding of [125I] labelled IGF-I was higher in women (7.6 +/- 0.7%) than in men (5.3 +/- 0.4%). No significant difference was observed in maximal specific binding of [125I] labelled insulin between prepubertal children (8.2 +/- 0.5%) and adults (7.2 +/- 0.7%). In both groups, competition by unlabelled insulin for [125I] labelled insulin binding gave 50% displacement for approximately 0.25 nM and IGF-I was about 80-fold less potent. Both IGF-I and insulin binding parameters were not significantly correlated with plasma hormone levels. In prepubertal children, the high-affinity IGF-I receptors number decreased with increasing high-affinity insulin receptors number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human umbilical vein endothelial cells express high affinity receptors for factor Xa

The binding of [¹²⁵I]-factor Xa to human umbilical vein endothelial cell (HUVEC) monolayers was studied. At 7°C, [¹²⁵I]-factor Xa bound to a single class of binding sites with a dissociation constant value of 6.6 ± 0.8 nM and a binding site density of 57,460 ± 5,200 sites/cell (n = 3). Association and dissociation kinetics were of a pseudo-first order and gave association and dissociation rate constant values of 0.15 × 10⁶ M^-1 s^-1 and 4.0 × 10^-4 s^-1, respectively. [¹²⁵I]-factor Xa binding was inhibited by factor Xa but was not affected by factor X, thrombin or monoclonal antibodies against factor V, antithrombin-III or tissue factor pathway inhibitor (TFPI) but was inhibited by an antibody specific for the effector cell protease receptor-1 (EPR-1), a well-known receptor of factor Xa on various cell types. [¹²⁵I]-factor Xa binding to HUVEC was not affected by various inhibitors of factor Xa such as DX 9065, pentasaccharide-antithrombin-III or TFPI. Factor Xa increased intracellular free calcium levels and phosphoinositide turnover in endothelial cells and, when added to HUVEC in culture, factor Xa was a potent mitogen, stimulating an increase in cell number at a 0.3 to 100 nM concentration. HUVEC-bound factor Xa promoted prothrombin activation in the presence of factor Va only. This effect was inhibited by both indirect and direct inhibitors of factor Xa. These findings indicate that HUVEC express functional high affinity receptors for factor Xa, related to EPR-1, which may be of importance in the regulation of coagulation and homeostasis of the vascular wall. J. Cell. Physiol. 172:36–43, 1997. © 1997 Wiley-Liss, Inc.     

Albumin absorption by canine bronchial epithelium

Albumin concentrations in airway surface liquid are low compared with plasma. To investigate the mechanisms that generate albumin gradients across airway epithelia, we have investigated whether active albumin absorption is a feature of bronchial epithelia. Freshly excised canine bronchi were mounted in Ussing chambers and short-circuited. Permeability coefficients of 14C-labeled canine albumin (Palb) were measured in the mucosal-to-submucosal (M----S) and submucosal-to-mucosal (S----M) directions in conductance-matched tissues. Mean steady-state values for Palb in the absorptive (M----S) direction (5.97 +/- 1.89 x 10(-7) cm/s) were significantly greater than rates in the S----M direction (1.09 +/- 0.41 x 10(-7) cm/s). Simultaneous measurements detected no asymmetry of transport of the fluid phase marker [3H]inulin. Gel filtration chromatography demonstrated that the majority of the radiolabel released into the submucosal bathing solution represented albumin fragments. Albumin fragments per se were not transported because no asymmetries in permeabilities of albumin fragments isolated from spontaneous degradation of tracer were detected. Decreasing the temperature of the bathing solution from 37 to 4 degrees C completely inhibited net albumin absorption. [14C]albumin transport was saturated by addition of high concentrations of unlabeled albumin (estimated Michaelis constant = 1.6 x 10(-3) M). These results demonstrate that albumin is absorbed by a low-affinity process that may contribute to the maintenance of low albumin concentrations in secretions.     

Characterization of the receptor for insulin-like growth factor II in bone cells

We have previously shown that insulin-like growth factor II (IGF-II) is produced by bone cells and that IGF-II stimulates cell proliferation and collagen synthesis in bone cells. We now extend these in vitro findings by demonstrating specific IGF-II binding to bone cells derived from newborn mouse calvaria and embryonic chick calvaria. The kinetics of [125I] IGF-II binding in embryonic chick calvaria cells showed time and temperature dependence. Scatchard analysis of [125I]IGF-II binding to chick calvaria cells showed an apparent Kd of 1.4 x 10(-10) M, with a calculated receptor site concentration of 40,000/cell. The specificity characteristics showed that IGF-II was significantly more potent than IGF-I or insulin in displacing IGF-II tracer. Competition for binding of [125I]IGF-II by unlabeled IGF-II showed a dose-dependent displacement between 0.5 and 25 ng/ml. Fifty percent displacement of [125I]IGF-II binding to chick and mouse calvarial cells was achieved at 1-2 ng/ml; 90% of specific binding of [125I]IGF-II was displaceable in the presence of 125 ng/ml of unlabeled IGF-II. IGF-I showed less than 5% cross reactivity for displacement of [125I]IGF-II binding to chick and mouse bone cells. Type II receptor inhibitory antibodies, R-II-PAB1 inhibited the binding of [125I]IGF-II to mouse bone cells and H-35 rat hepatoma cells (which contain type II but not type I receptors) in a dose-dependent manner. R-II-PAB1 also inhibited basal cell proliferation as well as IGF-II-, IGF-I-, and fibroblast growth factor (FGF)-induced cell proliferation in mouse bone cells. In chick calvaria bone cells and TE89 human osteosarcoma cells, R-II-PABI inhibited neither binding of [125I]IGF-II nor IGF-II-induced cell proliferation. These results together with our findings that IGF-II increased chick bone cell proliferation in the presence of maximal doses of IGF-I suggest that at least part of the mitogenic action of IGF-II is mediated through type II rather than type I receptors in bone cells.     

Thrombin binds to murine bone marrow-derived macrophages and enhances colony-stimulating factor-1-driven mitogenesis

The binding and mitogenic properties of thrombin have been established in various transformed cell lines. In such systems, thrombin induces cell division in the absence of exogenous growth factors, and the enzyme is considered to act directly as a mitogen. This study explores thrombin's interaction with nontransformed, growth factor-dependent cells. Binding of 125I-alpha-thrombin to colony-stimulating factor (CSF)-1-dependent bone marrow-derived macrophages is saturable, time-dependent, and displaceable by both unlabeled alpha-thrombin, and esterolytically inactive thrombin. Both dissociation studies of pre-bound radio-labeled thrombin and Scatchard analysis assisted by the program "Ligand" suggest adherence of thrombin-binding data to a multi-site model. There are an estimated 2 x 10(4) high affinity sites (Kd = 7 x 10(-9)M) and 2 x 10(6) low affinity sites (Kd = 9 x 10(-7)M) per cell. Quiescent bone marrow-derived macrophages were cultured with either 10(-8)M thrombin, 1000 units of CSF-1/ml, or both and [3H]thymidine incorporation was determined. Thrombin alone did not induce mitogenesis. CSF-1 induced mitogenesis with peak [3H] thymidine incorporation occurring 24 h after addition of the mitogen. This CSF-1-dependent mitogenic influence was enhanced greater than 2-fold by treatment with thrombin.     

A serum-free defined culture system which maintains follicle-stimulating hormone responsiveness and differentiation of porcine granulosa cells

A serum-free defined culture system has been developed that maintains follicle-stimulating hormone (FSH)-dependent differentiation of porcine granulosa cells from small follicles for up to six days in culture. Confluent monolayers of epithelioid cells were established after culture on fibronectin-coated culture dishes (FBN, 2 micrograms/cm2) in nutrient medium supplemented with human low-density lipoprotein (LDL, 10 micrograms/ml), insulin (I, 1 microgram/ml), and thrombin (TH, 1 NIH U/ml). Each of these factors was necessary to maintain the epithelioid morphology of the monolayers that attained 70% of the protein content and 71% of the cell number of replicate cultures maintained in nutrient medium supplemented with 10% fetal calf serum and insulin. Addition of FSH to the FBN/LDL/I/TH-supplemented cultures resulted in dose-dependent increases in progesterone secretion and [125I]-iodo-human chorionic gonadotropin (hCG) binding comparable to those obtained in the cultures containing serum. These results indicate that the attachment, epithelioid morphology, and differentiated function of porcine granulosa cells (GCs) can be maintained in defined culture conditions. This culture system will facilitate study of the effects of growth promoters and differentiative agents on GC function in the absence of poorly defined serum supplements.     

Role of albumin arginyl sites in albumin-induced reduction of endothelial hydraulic conductivity

We determined the effect of albumin on endothelial hydraulic conductivity (Lp) and the contributions of the positively charged arginyl and lysinyl residues of albumin in mediating the effect. Studies were made using monolayers of cultured sheep pulmonary artery endothelial cells grown to confluence on polycarbonate filters. Water flux was measured as transendothelial hydrostatic pressure was varied from 5 to 20 cm H2O. Lp was calculated from the slope of the relationship of water flux versus pressure. The Lp of endothelial monolayers perfused with albumin-free Hanks Balanced Salt Solution (HBSS) was compared to perfusion with HBSS containing either native albumin, or albumin in which the arginyl residues were modified by a condensation reaction with 1,2-cyclohexanedione (CHD-albumin), or albumin in which the lysinyl residues were modified by a substitution reaction with succinic anhydride (SC-albumin). Baseline Lp at 2.5 mg/ml native albumin was 1.6 +/- 0.1 X 10(-6) cm/s/cm H2O compared to the filter Lp after removing cells of 3.0 +/- 0.3 X 10(-4) cm/s/cm H2O. Endothelial Lp increased by 60% when albumin concentration was decreased from 2.5 mg/ml to 0.5 mg/ml (P less than 0.05), but did not change with an increase in concentration to 10 mg/ml. Albumin-free buffer and CHD-albumin increased endothelial Lp by 2.2 +/- 0.3-fold and 1.9 +/- 0.3-fold, respectively (P less than 0.05). All endothelial Lp values were restored to baseline when the native albumin concentration was returned to 2.5 mg/ml. Excess l-arginine (2 X 10(-3) M) inhibited the effect of native albumin and increased endothelial Lp 1.5 +/- 0.02-fold (P less than 0.05), but excess l-lysine (4 X 10(-3) in the presence of native albumin had no effect on Lp. None of the perfusates altered the filter Lp value. Neutral dextran (70 kD), in contrast to native albumin, had no effect on endothelial Lp. These results indicate that albumin reduces the hydraulic conductivity of endothelial monolayers in a concentration-dependent fashion and that the arginyl residues of albumin are required for the response. The effect of albumin may be mediated by a charge interaction of albumin with the endothelium.     

Phosphoinositide breakdown as an indirect link between stimulation and aggregation of rat platelets by thrombin and collagen

When washed rat platelets (1.5 x 10(9)/ml) were stimulated by a threshold concentration of thrombin (0.3 unit/ml) or collagen (10 micrograms/ml), a lag period of about 10 or 30 s, respectively, was seen before the start of aggregation. During the lag period, [32P]phosphatidylinositol 4,5-bisphosphate was degraded as the earliest event within 5-10 s of addition of the stimulus. However, though the extent of phosphatidylinositol 4,5-bisphosphate degradation within 10 s of addition of collagen was greater than that within 20 s of addition of thrombin (0.3 unit/ml), a lag of about 20 s remained before the initiation of aggregation by collagen. This casts doubt on the hypothesis that the stimulus-dependent phosphatidylinositol 4,5-bisphosphate breakdown induces the aggregation of platelets. Phosphatidylinositol labeled with 32Pi or [1-14C]arachidonic acid was scarcely degraded during the lag period. As aggregation proceeded, [14C-arachidonic acid]phosphatidylinositol was degraded with generation of diacylglycerol, phosphatidic acid, arachidonic acid and its metabolites. The maximum aggregation by collagen of rat platelets in which arachidonic acid of phospholipids was replaced in vivo with eicosapentaenoic acid was reduced, but that by thrombin was not, though reduction of thromboxane A2 generation was caused by both stimuli. Indomethacin also fully inhibited the aggregation induced by collagen, but not that induced by thrombin. Hence, thromboxane A2 is required for full aggregation by collagen, but not that by thrombin. These results indicate that thrombin-induced phosphoinositide metabolism may proceed independently of aggregation.     

Cytochalasin B-induced alterations of insulin binding and microfilament organization in cultured fibroblasts

The effect of cytochalasin B (CB) on insulin binding has been investigated in confluent cultures of chick embryo fibroblasts. Time- and dose-dependent increases in binding of [¹²⁵I]insulin was observed after incubation of fibroblasts with CB. At 10 μg/ml, CB caused a 2-fold increase in binding, due to an increase in the number of binding sites from 9.3 × 10³ to 2.0 × 10⁴ per cell. Removal of CB from the growth medium was accompanied by a decrease in [¹²⁵I]insulin binding to control values in 24 h. Increase in the binding of insulin in CB-treated CEF was also accompanied by enhancement of insulin to stimulation of [³H]thymidine incorporation into acid-insoluble material. CB treatment also caused disorganization and disappearance of microfilament bundles and changes in cell shape from flat, with a few blebs and folds on the cell surface, to round with numerous blebs and folds. The data from this study suggest that changes in the number of surface insulin-binding sites may be related to the state of organization of cytoskeletal structures in chick embryo fibroblasts.     

Characterization of a scintillation proximity assay to detect modulators of transforming growth factor alpha (TGF alpha) binding.

A scintillation proximity assay (SPA) for transforming growth factor alpha (TGF alpha) using SPA beads coated with A431 membranes has been studied. Binding of TGF alpha to the beads was characteristic of a receptor interaction. A class of high-affinity receptors for [125I]-TGF alpha (Kd = 0.10-0.26 nM) was detected by competition studies between [125I]TGF alpha and cold TGF alpha and by analysis of association and dissociation rate constants. An antibody to the epidermal growth factor receptor (clone 528) inhibited binding of [125I]TGF alpha (IC50 = 0.20 micrograms/ml), but an anti-TGF alpha antibody (clone 134A-2B3) (less than 25 micrograms/ml) did not block binding. Suramin inhibited [125I]-TGF alpha binding (IC50 = 0.20 mM). The ether lipids 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine, and rac-lyso-platelet activating factor inhibited TGF alpha binding (IC50 values of 49, 69, and 57 microM, respectively). SPA is a convenient method for identifying agents that may act by interfering with TGF alpha binding.     

Clearance of different-sized proteins from the alveolar space in humans and rabbits.

Investigation of the clearance of proteins from the air spaces is important for an understanding of the resolution of pulmonary edema and also because of current interest in delivery of therapeutic peptides via the distal air spaces. Few experimental studies have examined the size dependence for alveolar clearance of large macromolecules; there have been no human studies. In anesthetized rabbits, we measured clearance of cyanocobalamin and different-sized human proteins instilled into the air spaces. After 8 h, the amounts of instilled tracer recovered in the lungs were [57Co]cyanocobalamin, 19.4 +/- 3.0% (Stokes radius 0.65 nm); 125I-labeled insulin, 64.6 +/- 3.9% (1.2 nm); 131I-labeled albumin, 87.0 +/- 4.0% (3.5 nm); and 125I-labeled immunoglobulin G, 91.8 +/- 3.3% (5.5 nm) (P < 0.05). Sieving of different-sized proteins occurred across the alveolar epithelial barrier because tracer concentrations in air space lavage fluid after 8 h were decreased more for the smaller tracers than the larger ones. Size selectivity for alveolar protein clearance in humans with resolving alveolar edema was investigated by measuring the changes in albumin and total protein concentration. The fraction of total protein concentration made up of albumin was greater in the edema fluid than in the plasma initially. The albumin fraction decreased with time in 9 of 10 patients with resolving edema, from 0.62 +/- 0.2 to 0.58 +/- 0.10 (P < 0.05) after 10 +/- 5 h. Thus both rabbit studies and human studies provide evidence for size-dependent clearance of protein from the air spaces of the lung.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of GROalpha-induced human endothelial cell proliferation by the alpha-chemokine inhibitor antileukinate

GROalpha, an autocrine mitogenic factor for melanoma cell lines, belongs to the superfamily of alpha-chemokines. Here, we report that GROalpha stimulates the growth of human umbilical vein endothelial cells (HUVEC) in vitro, with proliferation being significantly stimulated by 100 nM recombinant human (rh) GROalpha. Proliferation was significantly inhibited by 100 microg/ml anti- human GROalpha monoclonal antibody (mAb), while excess GROalpha restored the growth. The addition of rhIL-8, rhIP-10, anti-human IL-8 or anti-human ENA-78 mAbs did not alter HUVEC proliferation. [125I]IL-8 binding to HUVEC was saturable and inhibited by non-radioactively iodinated IL-8, but not non-iodinated IL-8. [125I]GROalpha binding was also inhibited by iodinated IL-8. Since these data suggested specific binding sites for alpha-chemokines on HUVEC, we tested the effect of antileukinate, a potent alpha-chemokine receptor inhibitor, on [125I]GROalpha binding. Antileukinate inhibited GROalpha binding and suppressed HUVEC proliferation in a dose-dependent manner. Antileukinate was not cytotoxic, with no decrease in cell viability in the presence of 100 microM antileukinate. These findings suggest that GROalpha is essential for HUVEC growth factor and that antileukinate inhibits growth by preventing autocrine GROalpha receptor binding. This raises the interesting possibility of alpha-chemokine receptor inhibitors, such as antileukinate, in the treatment of cancer where angiogenesis is an important factor for tumour growth.     

Distribution of receptors for insulin and insulin-like growth factor I (somatomedin C) in the adrenal gland

Rat adrenal glands contain cell surface high-affinity receptors for several peptide hormones. Receptors for IGF-I were abundant in this tissue, but receptors for insulin were relatively scarce. The behavior of adrenal membrane IGF-I receptors in radioligand binding assays was similar to the behavior of IGF-I receptors from other tissues, with a KD congruent to 6.2 x 10(-9) M. Covalent cross-linking studies with [125I]IGF-I revealed an IGF-I receptor alpha-subunit with Mr congruent to 135,000 on dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, as well as a smaller radiolabeled peptide, Mr = 116,000. In contrast, little binding of [125I]insulin to adrenal membranes was observed and no labeling occurred in cross-linking studies using [125I]insulin. These results contrast with the findings of whole-body autoradiographic studies that indicated substantial binding of [125I]insulin to adrenal glands and suggest that IGF-I, rather than insulin, may play a critical role in the growth and development of the adrenal gland.     

Mouse glomerular endothelial cells have an insulin receptor

An insulin receptor was found on the surface of cloned mouse glomerular endothelial cells in vitro. Total specific binding was 2.5 +/- 0.3%/10(6) cells at 90 min and 22 degrees C. Analysis according to Scatchard resulted in a curvilinear plot, with a kd for the high and low affinity sites estimated at 1.41 x 10(-10) and 8.2 x 10(-8) respectively. Insulin binding decreased following 12 hour exposure to 50 ng/ml of insulin suggesting that down regulation of the receptor had occurred, an effect which was reversible. Covalent crosslinking of the receptor to 125I insulin revealed one band at Mr 125,000 by SDS-PAGE which disappeared following preincubation with excess unlabeled insulin. Insulin was also able to stimulate phosphorylation of the beta subunit. The characteristics of this insulin receptor appear very similar to that of endothelial cell types from other microvascular beds.     

Binding of thrombin to cultured human fibroblasts: evidence for receptor modulation

Previous work from this laboratory has indicated that thrombin's influence on cell growth can be negative as well as positive. Addition of enzyme to actively growing or confluent cultures of human skin fibroblasts produced growth stimulation, whereas cultures receiving thrombin at the time of subculture displayed inhibited DNA synthesis and mitosis. The specific binding of [125I]thrombin to cells under stimulatory and inhibitory conditions has been studied. Fibroblasts receiving enzyme at subculture bound about two times more [125I]thrombin than those processed in the same way several hours later. The apparent dissociation constant for both groups was approximately 1.5 x 10(-8) M. In each case binding was saturable, although cells receiving enzyme at subculture showed a much higher rate of binding. Experiments were conducted in which enzyme was added to cells at various times after subculture. It was found that the ability of these fibroblasts to specifically bind [125I]thrombin decreased progressively over a 2-h period after subculture and then remained constant for at least 24 h. Evidence is also presented indicating that the binding of [125I]thrombin in both experimental groups was inversely dependent upon the culture density. The biological effects of elevated thrombin binding in cells receiving enzyme at subculture were examined. It was found that inhibited DNA synthesis and altered cellular morphology were directly to this parameter. This study suggests that fibroblasts may possess cryptic thrombin receptors that become exposed during subculture or after injury in vivo. These possibilities and the relationship of cell shape to the availability of thrombin receptors are discussed.     

The expression of high molecular weight kininogen on human umbilical vein endothelial cells

High molecular weight kininogen (HMWK) functions as a cofactor for activation of plasma serine zymogens and as an inhibitor of tissue cysteine proteases. Cell surfaces to which HMWK binds may provide sites for regulation of these systems. Localization of these HMWK-dependent processes at sites of vascular injury may depend on its binding to specific receptors on endothelial cells. In culture, passaged human umbilical vein endothelial cells (HUVEC) bind anti-HMWK antibody to the cell surface and contain 171 +/- 75 ng of HMWK/10(8) cells. [35S]Methionine-labeled HUVEC in culture synthesize a 120-kDa protein immunoisolated using an anti-kininogen antibody, and a 3500-nucleotide message for human HMWK was detected by Northern blot in RNA extracted from HUVEC. HUVEC also express unoccupied binding sites for HMWK on their surface. 125I-HMWK specifically binds to HUVEC in a reaction requiring Zn2+. 125I-HMWK binding to HUVEC is saturable at 4 degrees C but not at 23 degrees C. 125I-HMWK binds to HUVEC with equal affinity as unlabeled HMWK. Kallikrein, factor XII, fibrinogen, fibronectin, and thrombin do not inhibit 125I-HMWK binding to HUVEC. 125I-HMWK-HUVEC binding remains fully reversible at 60 min following the addition of a 50-fold molar excess HMWK. HUVEC express 9.3 +/- 2.0 X 10(5) (mean +/- S.E.) HMWK binding sites/cell (Kd = 52 +/- 13 nM). Both added and cell-bound 125I-HMWK migrate at 120 kDa on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein remains uncleaved upon binding to the HUVEC surface. These studies indicate that HUVEC synthesize HMWK and the HUVEC surface has a site for its expression. By synthesizing and localizing HMWK to the cell surface, endothelial cells may contribute to the activation of plasma's contact serine zymogens and regulation of tissue cysteine proteases.     

The interaction of a glycosaminoglycan, heparin, with HIV-1 major envelope glycoprotein.

We demonstrate in vitro the occurrence of a specific but low-affinity interaction between soluble tetrameric rgp160 or soluble monomeric or tetrameric rgp120 and heparin-agarose (HA). This interaction is saturable, pH and temperature-dependent, and can be inhibited by soluble heparin, but not by soluble dextran. In buffer supplemented with 10 mM CaCl2, the C50 of soluble heparin, i.e., the concentration of soluble heparin which leads to 50% inhibition of the binding of [125I]rgp160 or of [125I]rgp120 to HA, is 1.1 x 10(-4) disaccharidic molar concentration for rgp160 and 3.2 x 10(-4) dissacharidic molar concentration for rgp120, which indicates low-affinity interactions. Upon chromatography on HA, [125I]rgp160 is repeatedly eluted as a retarded fraction when compared to the elution volume of [125I]rgp160-soluble heparin complex. Under the same experimental conditions, [125I]rgp120 is also eluted, but as a less retarded fraction than [125I]rgp160. Taken together, these results suggest that, at least part of the described anti HIV-1 activity of heparin might be mediated by interaction with HIV-1 major envelope glycoprotein.     

Radioautographic and quantitative study of insulin binding sites in the rat brain

In the present study, we describe the specificity and the autoradiographic distribution of insulin binding sites in the rat central nervous system (CNS) after in vitro incubation of brain sections with [125I]-14A insulin. Increasing concentrations of unlabeled insulin produced a dose-dependent inhibition of [125I]-insulin binding which represented 92 +/- 2% displacement with 3 X 10(-5) M, whatever the brain sections tested. Half-maximum inhibition with native insulin was obtained with 2.2 X 10(-9) M, with 10(-7) M proinsulin whereas glucagon had no effect. Under our experimental conditions, no degradation of [125I]-insulin was observed. Autoradiograms obtained by apposition of LKB 3H-Ultrofilm showed a widespread distribution of [125I]-insulin in rat CNS. However, quantitative analysis of the autoradiograms with 10(-10) M of labeled insulin, showed a high number of [125I]-insulin binding sites in the choroid plexus, olfactory areas, in both cerebral and cerebellar cortices, the amygdaloid complex and in the septum. In the hippocampal formation, the dorsal dentate gyrus and various subfields of CA1, CA2 and CA3 were labeled. Moreover, arcuate, dorso- and ventromedial nuclei of the hypothalamus contained high concentrations of [125I]-insulin whereas a low density was observed in the mesencephalon. The metabolic role of insulin in the CNS is supported by the large distribution of insulin binding sites in the rat brain. However, the presence of high affinity binding sites in selective areas involved in perception and integrative processes as well as in the regulation of both feeding behavior and neuroendocrine functions, suggests a neuromodulatory role of insulin in the brain.     

Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.