Characterization of endothelin receptors ETA and ETB expressed in COS cells.

The cloned cDNA genes for endothelin receptors ETA and ETB were expressed in COS cells, and the binding characteristics of the two receptors with three isopeptide ligands (ET-1, ET-2, and ET-3) were examined in detail. The results indicated that the stability of receptor-ET-1 complexes formed with ETA and ETB were significantly different from each other, while their affinities to ET-1 were similar. The preformed ETA-ET-1 complex readily dissociated upon SDS-PAGE, as did many of the other receptors so far studied, while the ETB-ET-1 complex survived SDS-PAGE when it was run at low temperature (approximately 4 degrees C). Clear differences in stability were also shown in comparative studies of acid treatment of the two types of complexes. Only the ETB-ET-1 complex was resistant to acid treatment (0.2 M acetic acid, 0.5 M NaCl), and its 50 kDa monomeric receptor-ligand complex remained intact. The ETB-ET-1 complex (50 kDa) formed at 4 degrees C on the surface of COS cells, however, was susceptible to limited proteolysis at 37 degrees C that reduced the molecular size of the complex to a distinct 35 kDa. No such size reduction was observed with the preformed ETA-ET-1 complex. The overall structure of two endothelin receptors, as deduced from the sequence of cloned cDNAs, is similar in many respects. However, the present findings demonstrate distinct differences in the biochemical nature of the two receptors, which suggest their distinct biological functions.     

Biochemical properties of rat protein kinase C-eta expressed in COS cells.

Using a PKC-epsilon cDNA probe a cDNA for PKC-eta has been cloned from a rat lung cDNA library. When expressed in COS cells, rat PKC-eta appeared as an 84 kDa protein. PKC-eta expressed in COS cells, was solubilized by 1% Triton X-100 and purified away from the endogenous PKC-alpha by ammonium sulphate fractionation. The activity of this PKC-eta preparation was characterized with respect to cofactor dependence and substrate specificity. Various PKC pseudosubstrate peptides are phosphorylated by PKC-eta in a phospholipid and TPA-dependent but calcium-independent manner. The polypeptide histone IIIS is a poor substrate.     

The binding of fucose-containing glycoproteins by hepatic lectins. The binding specificity of the rat liver fucose lectin

The parameters that affect the interaction of ligands with a fucose-binding lectin from rat liver have been examined. 125I-Fucosyl-bovine serum albumin (Fuc-BSA) containing 50 residues of fucose/molecule was used as the standard ligand. At low initial concentrations of ligand (10 ng/ml) and lectin (140 ng/ml), the reaction reaches equilibrium at pH 7.8, 23 degrees C, within 40 min. The binding of ligands is Ca2+ dependent with half-maximal binding occurring at 54 microM Ca2+; of several metal ions tested, only Sr2+ partially replaced Ca2+. Binding was maximal between pH 7.6 and 8.6, fell slightly up to pH 10, but fell markedly below pH 7. The lectin-ligand complexes dissociated at low pH, on removal of Ca2+, or in the presence of a large excess of competing ligand. The apparent association constant (Ka) for Fuc-BSA was 1.75 X 10(8) M-1. The fucose content of the Fuc-BSA also influenced binding, with little apparent binding below 24 fucose residues/molecule and maximal binding from 40 to 50 fucose residues/molecule. With knowledge of the parameters influencing binding, sensitive reproducible assays for the lectin were developed. The binding specificity of the lectin was examined by measuring the inhibition of 125I-Fuc-BSA binding by neoglycoproteins, monosaccharides, and glycosides or by direct binding of neoglycoproteins. Galactosides and beta-linked fucosides were the best ligands among the neoglycoproteins, with much weaker binding by mannosyl- or N-acetylglucosaminyl-BSA. On the basis of the pattern of inhibition of Fuc-BSA binding by various monosaccharides and glycosides, it is possible to propose the conformations of saccharides that best fit the lectin-binding site. The C1 conformation of N-acetyl-D-galactosamine fits best, although other not obviously related monosaccharides such as L-fucose, L-arabinose, and D-mannose can also assume conformations that permit them to be effective inhibitors. The pattern of binding of neoglycoproteins to the lectin differs from that of other pure hepatic lectins. Thus, the fucose lectin has a high affinity for Fuc-BSA and galactosyl-BSA but a low affinity for N-acetylglucosaminyl-BSA. The galactose lectin binds only galactosyl-BSA and shows little binding with either N-acetylglucosaminyl-BSA or Fuc-BSA. In contrast, the mannose/N-acetylglucosamine lectin binds N-acetylglucosaminyl-BSA and Fuc-BSA but not galactosyl-BSA.     

Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells

We used transient transfection in COS cells to compare the properties of mouse muscle acetylcholine receptors (AChRs) containing alpha, beta, delta, and either gamma or epsilon subunits. gamma- and epsilon-AChRs had identical association rates for binding 125I-alpha-bungarotoxin, and identical curves for inhibition of toxin binding by d-tubocurarine, but epsilon-AChRs had a significantly longer half-time of turnover in the membrane than gamma-AChRs. A myasthenic serum specific for the embryonic form of the AChR reduced toxin binding to gamma-, but not epsilon-AChRs. The gamma-AChRs had channel characteristics of embryonic AChRs, whereas the major class of epsilon-AChR channels had the characteristics of adult AChRs. Two minor channel classes with smaller conductances were also seen with epsilon-AChR. Thus, some, but not all, of the differences between AChRs at adult endplates and those in the extrasynaptic membrane can be explained by the difference in subunit composition of gamma- and epsilon-AChRs.     

Regulation of platelet-activating factor receptors in rat Kupffer cells

Ligand binding studies indicate that 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) down-regulates its own receptors on the plasma membrane of isolated rat Kupffer cells but has no significant effect on the binding affinity of the receptor for AGEPC. Exposure of isolated rat Kupffer cells to 10(-8) and 10(-6) M AGEPC resulted in a rapid, time-dependent reduction in the number of cell surface AGEPC receptors to a new steady state concentration (54.1 +/- 5.0% and 38.6 +/- 5.4% of control, respectively). During the observation period (6 h), the half-time of surface AGEPC receptors was about 60 and 45 min in the presence of 10(-8) and 10(-6) M AGEPC, respectively. Both the rate of loss and the maximal loss of the receptors were dependent upon the AGEPC concentration. With receptor synthesis inhibited by cycloheximide in the absence of AGEPC, the half-time of the surface AGEPC receptor was about 4 h, suggesting that AGEPC receptors are not recycled and that the loss of AGEPC receptors from the plasma membrane is accelerated by AGEPC binding. When incubated with Kupffer cells at 37 degrees C for 3 h, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (1.0 microM), an inactive metabolite of AGEPC, did not cause the loss of AGEPC receptors. Under the same conditions, AGEPC antagonists such as BN52021 (2 x 10(-5) M) or U66985 (2 x 10(-5) M) alone had no effect (97.0 +/- 3.9% of control for BN52021) or only a relatively slight effect (78.4 +/- 1.8% for U66985) on the number of surface AGEPC receptors. However, AGEPC antagonists inhibited the AGEPC-induced down-regulation of AGEPC receptors in a concentration-dependent manner, suggesting that the AGEPC-induced down-regulation of AGEPC receptors is a receptor-mediated process. The AGEPC-mediated decrease in receptor number on rat Kupffer cells is reversible. Upon removing AGEPC from the culture medium, about 67% of the lost receptors were replaced within 2 h. Cycloheximide, an inhibitor of protein synthesis, prevented the restoration of the AGEPC receptors. Similar results were obtained when Kupffer cells were incubated with Pronase followed by removing Pronase and reincubating the cells with or without cycloheximide. These observations suggest that the restored AGEPC receptor is newly synthesized rather than recycled. The present study demonstrates that under non-stimulatory (i.e. in the absence of AGEPC) conditions AGEPC receptors are lost from the plasma membrane and are reformed in the cells continuously.(ABSTRACT TRUNCATED AT 400 WORDS)     

The ligand binding specificity and tissue localization of a rat alveolar macrophage lectin

The parameters of the reaction between a rat alveolar macrophage lectin (Mr = 180,000) and its ligands have been examined. The reaction is dependent on Ca2+ over the optimal pH range for binding. The apparent dissociation constant for fucosyl bovine serum albumin, the standard ligand used in these studies, is 1.4 X 10(-10) M. The ligand binding specificity was determined by measurement of the inhibition of binding of fucosyl bovine serum albumin by various glycoproteins and saccharides. D-Mannose, L-fucose, and N-acetyl-D-glucosamine were the most effective inhibitors, and D-galactose was much poorer. The equatorial hydroxyl groups on the C-3 and C-4 of the mannose ring are important in the lectin-ligand interaction, and the axial hydroxyl group on the C-2 contributes to a lesser extent. Immunocytological studies revealed that the lectin isolated from alveolar macrophages is widely distributed in other rat tissues. Hepatocytes are devoid of the lectin, but hepatic Kupffer cells and endothelial cells contain significant amounts. This was confirmed by isolation of the lectin from liver. Spleen and skeletal muscle also contain lectin, but much smaller amounts were found in brain, kidney, and heart muscle.     

Carbohydrate binding specificity of a beetle (Allomyrina dichotoma) lectin

Carbohydrate binding specificity of a lectin, allo A, isolated from a beetle (Allomyrina dichotoma), was investigated by means of lectin affinity chromatography. Sialylated complex-type and hybrid-type oligosaccharides/glycopeptides, and sialyllactose were retained by the column, whereas desialylated ones were retarded but not retained by the column. The association constants of allo A for biantennary oligosaccharides from human serum transferrin, determined by frontal analysis, were 8.0 X 10(5) M-1, 4.5 X 10(5) M-1, and 2.5 X 10(5) M-1 for disialo-, monosialo-, and asialo-oligosaccharides, respectively. Removal of the beta-galactose residues markedly reduced the association constant to 3.5 X 10(3) M-1. Furthermore, allo A was found to have no affinity for mucin-type glycopeptides carrying the sialylated Gal beta 1----3 GalNAc sugar sequence (Ka: 3.5 X 10(3) M-1). The results of this study indicated that allo A strongly binds to the trisaccharide structure, NeuAc alpha 2-3(6)Gal-beta 1-4GlcNAc, and that its binding potency is affected by the inner core structures of oligosaccharides and glycopeptides, because the presence of a bisecting N-acetyl-glucosamine residue and an alpha-fucose residue linked to the innermost N-acetylglucosamine residue reduced the association constants for oligosaccharides and glycopeptides.     

The cellular specificity of lectin binding in the kidney

Summary In order to estimate the usefulness of lectins in the study of the functional segmentation of the nephron, the sites of binding of five lectins were identified in the rat kidney. Lectin-peroxidase conjugates were applied to cryostat sections. The bound conjugates were stained with 3,3-diaminobenzidine for light microscopical observation. Each lectin has a specific binding pattern along the nephron. Reversely, the different segments of the nephron defined by other histological methods can be identified on the basis of their affinity for lectins. The different parts of the thick ascending limb, namely the medullary segment, the cortical segment and the macula densa, can be distinguished even more readily with lectin histochemistry than with any other histochemical procedure. The binding of lectins to luminal membranes in some segments indicate the possibility to use lectins for the separation of particular cell types and for the modification of the transport properties of their membranes.This research has been supported by the Swiss National Science Foundation, Grant No. 3.900-0.79     

Identification of receptors for platelet-activating factor in rat Kupffer cells

Ligand binding studies demonstrated that isolated rat Kupffer cells possess high affinity binding sites for platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC). AGEPC binding reached saturation within 10 min at 25 degrees C and was reversible. A Scatchard analysis revealed a single class of AGEPC receptors numbering about 10,600 sites/cell and possessing a dissociation constant of 0.45 nM. Similar values for the dissociation constant for AGEPC (0.12 and 0.34 nM) were obtained independently by kinetic analysis of specific AGEPC binding. AGEPC binding was stereospecific and was inhibited by Zn2+ and AGEPC receptor antagonists including BN52021 and U66985. The AGEPC receptor was functionally active since it was shown to mediate arachidonic acid release and eicosanoid production in Kupffer cells, and these events were inhibited by AGEPC receptor antagonist BN52021. The receptor-mediated arachidonic acid release was extracellular calcium-dependent and was abolished by calcium channel blocker prenylamine and by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, indicating that calcium influx through a receptor-regulated calcium channel in the plasma membrane is involved in the AGEPC-induced arachidonic acid release. It is suggested that rat Kupffer cells have specific and functionally active AGEPC receptors which are involved in signaling mechanisms which govern the production of several other autacoid-type mediators in the liver.     

Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-alpha in macrophage cell line and rat Kupffer cells

Recent studies have demonstrated that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis. However, the mechanism responsible for the beneficial effect of AM/AMBP-1 remains unknown. The aim of this study therefore was to determine whether AM/AMBP-1 directly reduces lipopolysaccharide (LPS)-induced secretion of TNF-alpha from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from normal rats. TNF-alpha release and gene expression were determined by ELISA and RT-PCR, respectively. The results indicated that LPS increased TNF-alpha production from RAW cells by 38-63-fold in a dose- and time-dependent manner. Although incubation with AM or AMBP-1 alone inhibited LPS-induced TNF-alpha release by 14-22% and 13-22%, respectively, AM and AMBP-1 in combination significantly suppressed TNF-alpha production (by 24-35%). Moreover, the upregulated TNF-alpha mRNA by LPS stimulation was significantly reduced by AM/AMBP-1, but not by AM or AMBP-1 alone. In the Kupffer cells primary culture, AM or AMBP-1 alone inhibited LPS-induced TNF-alpha production by 52% and 44%, respectively. Co-culture with AM/AMBP-1 markedly reduced TNF-alpha production (by 90%). Moreover, AM or AMBP-1 alone decreased TNF-alpha mRNA expression by 41% and 36%, respectively, whereas the combination of AM/AMBP-1 decreased its expression by 63%. These results indicate that AM and AMBP-1 in combination effectively suppress LPS-induced TNF-alpha expression and release especially from primary cultured Kupffer cells, suggesting that the downregulatory effect of AM/AMBP-1 on proinflammatory cytokine TNF-alpha may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.     

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.     

The distribution of circulating receptors on COS 7 cells

Rapidly circulating receptors on the surfaces of migrating or spreading cells are often concentrated toward the cells' leading edges. This polar distribution is the principal evidence that circulating membrane is returned to the surface of these cells at their fronts, where it is proposed to assist the forward extension of the cell. However, when low-density lipoprotein (LDL) receptors are transiently expressed in COS cells-cells which actively spread-their distribution is random, indicating that COS cells do not behave like other motile cells. This anomaly is examined here. I find that COS cell lines expressing either transferrin or LDL receptors do, in fact, usually have polar distributions of both receptors. The transient expression of LDL receptors interferes, in an unknown way, with the polar properties of COS cells. Furthermore, it is shown that whether receptors are located toward the cell's edge or are randomly distributed depends both on the ability of the receptor to circulate and on the state of the particular cell at that time.     

Fc receptors mediate prostaglandin and superoxide synthesis in cultured rat Kupffer cells

Latex beads with covalently bound bovine serum albumin were prepared and coated with anti-BSA immunoglobulin G. These particles were shown to possess on their surfaces a defined quantity of the antibody with the Fc portions exposed to the medium. One homologous and two heterologous antibodies of the G class were used and compared in terms of their binding to the rat Kupffer cells and their ability to elicit the typical phagocytotic responses. These particles were phagocytosed by rat Kupffer cells and elicited synthesis of prostaglandins and superoxide anion radicals. A significant release of superoxide into the medium was observed in the presence of cytochalasin B only. The data presented here suggest that a) Fc-carrying particles can be bound to Kupffer cells and elicit responses via specific receptors; b) coating with the homologous antibody yields the most effective particles; c) superoxide release into the surrounding medium is most abundant when the particle-binding membrane areas are prevented from forming phagocytotic vesicles.     

Biosynthesis in vitro and the pattern of lectin binding receptors in monkey kidney cell surfaces.

The glycosyltransferase activities involved in the biosynthesis $\text{in vitro}$ of neutral blood group-related glycosphingolipids were measured in African green monkey kidney cells (Vero) grown in culture. The a-fucosyltransferases which catalyzed the reaction between GDP-fucose and corresponding acceptors to form H-active and novel Le^a-type glycosphingolipids were characterized in membrane fractions isolated from Vero cells and monkey bone marrow. Using ¹²⁵I-labeled $\text{Ulex europeus}$ and $\text{Lotus tetragonolobus}$ lectins the differential binding to Vero cell surface glycoproteins and glycolipids was studied under various conditions.     

Neutralization of poliovirus by cell receptors expressed in insect cells.

To examine the interaction of the poliovirus receptor (PVR) with virus and the role of the PVR in virus entry, the PVR was expressed in insect cells. Poliovirus bound to insect cells infected with a recombinant baculovirus (AcPVR) carrying cDNA encoding the PVR. Antibodies raised against PVR expressed in bacteria immunoprecipitated a 67-kilodalton polypeptide from cytoplasmic extracts of AcPVR-infected cells. Treatment of AcPVR-infected cells with tunicamycin revealed that the PVR is a glycoprotein containing N-glycosidic linkages and that carbohydrate accounts for nearly 50% of its molecular weight as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When PVR was solubilized from AcPVR-infected insect cells and incubated with poliovirus, viral infectivity was neutralized. Sedimentation analysis revealed that irreversibly altered 135S particles were formed after incubation of poliovirus at 37 degrees C with solubilized extracts of AcPVR-infected insect cells. These results demonstrate that poliovirus eclipse may result from interaction with the cell receptor at neutral pH in the absence of membranes and suggest that soluble receptors may be effective antiviral agents against picornaviruses.     

Pharmacological characterisation of (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortropane (PE2I) binding to the rat neuronal dopamine transporter expressed in COS cells.

The interaction of (E)-N-(3-iodoprop-2-enyl)-2beta-Carbomethoxy-3beta-(4'-methylphenyl) nortropane (PE2I) with the rat neuronal dopamine transporter (DAT) was studied in transfected COS cells by measuring its ability to inhibit DA uptake and by measuring its affinity in radioligand binding experiments. Saturable [3H]DA uptake was measured in COS cells transiently transfected with the cDNA sequence encoding the rat DAT. Pharmacological characterisation of this uptake revealed functional properties with a V(max) value of 45.05+/-2.62 pmol/mg protein per min and a K(m) value of 2.86+/-0.28 microM. The specific [3H]DA uptake was fully inhibited by 1 microM PE2I. Concentration response curves revealed the high potency of PE2I in inhibiting DA uptake (pEC(50) value of 8.70+/-0.33), 25 times higher than that observed for the reference DAT inhibitor, GBR 12935. On crude homogenates from transfected COS cells, PE2I displaced the specific binding of [3H]GBR 12935 with a pK(i) value of 7.73+/-0.13. Accordingly, [125I]PE2I was found to specifically recognise a single binding site population which is almost completely displaced by GBR 12935 and nomifensine. Saturation experiments revealed the high affinity of [125I]PE2I (K(D) value of 3.8+/-0.63 nM) that correlates with the high potency of PE2I in inhibiting the [3H]DA uptake. This contrasts with the results obtained with GBR 12935 for which a discrepancy was found between its high affinity in binding assays (K(D) value of 0.43+/-0.04 nM) and its rather low potency in functional assays (pEC(50) value of 7.30+/-0.05). A relatively high level of [3H]GBR 12935 binding was detected in non transfected COS cells. Such nomifensine resistant binding is attributed to the interaction of GBR 12935 with cytochrome P-450 as it was displaced by cis-(Z)-flupentixol (an inhibitor of cytochrome P-450). Such interaction was not observed using PE2I. Taken together, these data demonstrate that PE2I was a highly potent inhibitor of cloned DAT compared with GBR 12935 and provided a useful tool for further investigations in cells transfected with cDNA encoding the DAT.     

The fate of lipopolysaccharide in cultured rat Kupffer cells

Cultured rat Kupffer cells were incubated in presence of biologically tritiated Salmonella abortus equi lipopolysaccharide. Uptake of lipopolysaccharide increased rapidly during the first 2 h of incubation and then levelled off. Within the first h of incubation 10(6) Kupffer cells were able to ingest up to 18 micrograms lipopolysaccharide. Kupffer cells metabolised lipopolysaccharide and released lipopolysaccharide-related substances, but neither the cell-associated lipopolysaccharide nor the released lipopolysaccharide products were detoxified, as measured by the mouse lethality test.     

The roles of mast cells and Kupffer cells in rat systemic anaphylaxis

Mast cells and other cells such as macrophages have been shown to mediate systemic anaphylaxis. We determined the roles of mast cells and Kupffer cells in hepatic and systemic anaphylaxis of rats. Roles of mast cells were examined by using the mast cell-deficient white spotting (Ws/Ws) rat; the Ws/Ws and wild type (+/+) rats were sensitized with ovalbumin (1 mg). Roles of Kupffer cells were examined by depleting Kupffer cells using gadolinium chloride or liposome-encapsulated dichloromethylene diphosphonate in the Ws/Ws and Sprague-Dawley rats. An intravenous injection of 0.6 mg ovalbumin caused substantial anaphylactic hypotension in both the Ws/Ws and +/+ rats; however, the occurrence was delayed in the Ws/Ws rats. After antigen, portal venous pressure increased by 13.1 cmH2O in the +/+ rats, while it increased only by 5.7 cmH2O in the Ws/Ws rats. In response to antigen, the isolated perfused liver of the Ws/Ws rats also showed weak venoconstriction, the magnitude of which was one tenth as large as that of the +/+ rats, indicating that hepatic anaphylaxis was primarily due to mast cells. In contrast, Kupffer cell depletion did not attenuate anaphylactic hepatic venoconstriction in isolated perfused livers. In conclusion, mast cells are involved mainly in anaphylactic hepatic presinusoidal portal venoconstriction but only in the early stage of anaphylactic systemic hypotension in rats. Macrophages, including Kupffer cells, do not participate in rat hepatic anaphylactic venoconstriction.