Radiolabeled Ligand Binding Research Articles

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabeled ligand binding techniques, we show that the binding of [3H]-(±)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15nM and 1μM. These classes of sites exist in approximately equal numbers and all [3H]-(±)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.

Pharmacological and immunological identification of native α7 nicotinic receptors: Evidence for homomeric and heteromeric α7 receptors

Controversy surrounds the expression of α7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, α7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [ 125I]α-bungarotoxin (αBGT) binding reaches equilibrium within 4 h and is saturable with a K d value of 4.2 nM. Using homologous competition experiments, the K i for binding of αBGT was 1.9 nM. These data are consistent with the expression of homomeric α7 nAChRs. Methyllycaconatine (MLA), which binds α7 nAChRs with high affinity, inhibits [ 125I]αBGT binding in a concentration-dependent manner with a K i of 30.6 nM; this value is ∼ 10 fold higher than the reported affinity of MLA for α7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate α7 nAChRs, as has been previous demonstrated for bovine adrenal α3β4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb319, an antibody which recognizes α7 nAChR protein, and then probed with mAb319 using Western blot analysis, a single band of ∼ 53 kDa is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes α3 and α5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of ∼ 53 kDa is identified. Together, these results support the expression of α7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric α7 nAChRs.

Novel Estrogen Receptor-Related Transcripts in Marisa cornuarietis; a Freshwater Snail with Reported Sensitivity to Estrogenic Chemicals

We have isolated novel molluskan steroid receptor transcripts orthologous to vertebrate estrogen receptors (ERs) and estrogen receptor-related receptors (ERRs) from the freshwater snail Marisa cornuarietis. Radiolabeled ligand binding analyses showed that neither recombinant receptor protein specifically bound 17beta-estradiol over the range applied (0.3-9.6 nM). These novel receptor transcripts have thus been designated mcER-like and mcERR respectively. Quantitative PCR revealed mcER-like to be expressed ubiquitously throughout a range of male and female structures studied, including neural and reproductive tissues. Highest absolute levels were seen in the male penis-sheath complex. The mcERR mRNA was also expressed ubiquitously throughout all male and female tissues analyzed here, with very low absolute transcript numbers in female accessory sex structures compared to other tissues.

In vivo and in vitro measurement of brain phosphodiesterase 4 in rats after antidepressant administration

Based largely on in vitro measurements, the mechanism of several antidepressant treatments is thought to involve upregulation of 3'-5'-cyclic adenosine monophosphate (cAMP) signal transduction cascade and a corresponding increase in phosphodiesterase (PDE) 4, the enzyme that metabolizes cAMP. To assess the in vivo status of PDE4, rats were chronically treated with imipramine and then studied with: (1) in vivo positron emission tomography (PET) measurement of (R)-[(11)C]rolipram binding, (2) in vitro measurement of [(3)H]rolipram binding in brain homogenates, and (3) Western blotting for protein levels of PDE4 isoforms. Imipramine administration caused no significant change in B(max)/K(d), for both in vivo measurements with (R)-[(11)C]rolipram and in vitro measurements with [(3)H]rolipram in frontal cortex, hippocampus, and diencephalon. None of 10 isoforms of PDE4A, B, and D measured with immunoblots of frontal cortex and hippocampus showed a significant change. In summary, using relatively large brain regions for both in vivo imaging and in vitro measures of radiolabeled ligand binding and protein levels, chronic imipramine treatment via continuous mini-pump administration caused no significant change in PDE4 levels. Most, but not all, prior in vitro studies have found increased PDE4 levels after antidepressant administration. The current results raise questions about the in vivo effects of antidepressant treatment on PDE4 and on other potentially important experimental factors (e.g., continuous infusion vs. intermittent injection of antidepressant) in large brain areas. However, the results do not deny possibility of changes in discrete areas, which were not studied in the current study applying PET.

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.

Agonist-stimulated β-Adrenergic Receptor Internalization Requires Dynamic Cytoskeletal Actin Turnover

Stimulation of beta-adrenergic receptors (betaARs) leads to sequential recruitment of beta-arrestin, AP-2 adaptor protein, clathrin, and dynamin to the receptor complex, resulting in endocytosis. Whether a dynamic actin cytoskeleton is required for betaAR endocytosis is not known. In this study, we have used beta(1)- and beta(2) ARs, two ubiquitously expressed members of the betaAR family, to comprehensively evaluate the requirement of the actin cytoskeleton in receptor internalization. The integrity of the actin cytoskeleton was manipulated with the agent latrunculin B (LB) and mutants of cofilin to depolymerize actin filaments. Treatment of cells with LB resulted in dose-dependent depolymerization of the cortical actin cytoskeleton that was associated with significant attenuation in internalization of beta(2)ARs, beta(1)ARs, and mutants of beta(1)ARs that internalize via either clathrin- or caveolin-dependent pathways. Importantly, LB treatment did not inhibit beta-arrestin translocation or dynamin recruitment to the agonist-stimulated receptor. To unequivocally demonstrate the requirement of the actin cytoskeleton for beta(2)AR endocytosis, we used an actin-binding protein cofilin that biochemically depolymerizes and severs actin filaments. Isoproterenol-mediated internalization of beta(2)AR was completely blocked in the presence of wild type cofilin, which could be rescued by a mutant of cofilin that mimics a constitutive phosphorylated state and leads to normal agonist-stimulated beta(2)AR endocytosis. Finally, treatment with jasplakinolide, an inhibitor of actin turnover, resulted in dose-dependent inhibition of beta(2)AR internalization, suggesting that turnover of actin filaments at the receptor complex is required for endocytosis. Taken together, these data demonstrate that intact and functional dynamic actin cytoskeleton is required for normal betaAR internalization.

Estrogen Receptor Testing of Breast Cancer in Current Clinical Practice: What's the Question?

Estrogen Receptor Testing of Breast Cancer in Current Clinical Practice: What's the Question?

Cell-specific Expression ofN-Methyl-D-Aspartate Receptor Subunits in Müller Glia and Neurons from the Chick Retina

Functional N-methyl-D-aspartate (NMDA) receptors (NMDARs) in Müller glia may influence glutamate neurotransmission through feedback loops between glia and neurons. The physiologic properties of NMDARs derive from the subunit composition of the tetrameric receptor. We characterized glycine binding to the NMDAR coagonist site in Müller cell membranes and determined NMDAR subunit expression in chick Müller glia compared with retinal neurons to make predictions about the heteromeric assembly of NMDARs. Kinetic and pharmacologic properties of the glycine coagonist site were determined by radiolabeled ligand binding to membrane preparations from chick Müller glia and retinal neurons in primary culture. The molecular composition of NMDARs was analyzed by RT-PCR amplification and Western blotting. The NMDAR coagonist site in Müller cell membranes has 5-fold lower affinity for glycine and 30-fold lower affinity for D-serine compared with values obtained for synaptic membranes from whole retina and with reported values in brain tissue. NR1 subunit N-terminal and C-terminal splice-variant expression also differs in Müller cells and retinal neurons. Pharmacologic characteristics of NMDAR coagonist-site differ in Müller glia and neurons from the retina, in agreement with the distinct subunit expression profile found. Whereas NMDARs in Müller glia contain exclusively exon 5 that lacks NR1 subunits, receptors in distinct subtypes of neurons may contain NR1 with or without exon 5, suggesting a cell-specific assembly of the NMDAR complex. Structural differences in NMDARs could underlie the differential participation of neurons and glia in the physiologic control of glutamate transmission in the retina.

Design and synthesis of labeled analogs of PhTX-56, a potent and selective AMPA receptor antagonist

Polyamines and polyamine toxins are biologically important molecules, having modulatory effects on nucleotides and proteins. The wasp toxin, philanthotoxin-433 (PhTX-433), is a non-selective and uncompetitive antagonist of ionotropic receptors, such as ionotropic glutamate receptors and nicotinic acetylcholine receptors. Polyamine toxins are used for the characterization of subtypes of ionotropic glutamate receptors, the Ca 2+-permeable AMPA and kainate receptors. A derivative of the native polyamine toxin, philanthotoxin-56 (PhTX-56), has recently been shown to be an exceptionally potent and selective antagonist of Ca 2+-permeable AMPA receptors. PhTX-56 and its labeled derivatives are promising tools for structure–function studies of the ion channel of the AMPA receptor. We now describe the design and synthesis of 3H-, 13C-, and 15N-labeled derivatives of PhTX-56 for molecular level studies of AMPA receptors. [ 3H]PhTX-56 was prepared from a diiodo-precursor with high specific radioactivity, providing the first radiolabeled ligand binding to the pore-forming part of AMPA receptors. For advanced biological NMR studies, 13C and 15N-labeled PhTX-56 were synthesized using solid-phase synthesis. These analogs can provide detailed information on the ligand–receptor interaction. In conclusion, synthesis of labeled derivatives of PhTX-56 provides important tools for future studies of the pore-forming region of AMPA receptors.

Détection des ciguatoxines : avantages et inconvénients des différentes méthodes biologiques utilisées

Ciguatera is a seafood intoxication that results from ingestion of reef fish contaminated with ciguatoxins at levels orally toxic for humans. Precursors of those toxins, gambiertoxins, are produced by benthic dinoflagellates (genus Gambierdiscus), and then accumulated and biotransformed by herbivorous and carnivorous fishes into ciguatoxins, more toxic for humans. In the absence of specific treatment, that disease remains a health problem with otherwise adverse socio-economic impacts. Thus a cost-effective means of detecting ciguatoxins in fish has long been searched for. Many assays have been developed, including in vivo, in vitro, chemical or immunochemical approaches. This review focuses on some biological methods, from the well-standardised mouse assay to the specific radio-labelled ligand binding assay that is performed on rat brain synaptosomes. In addition to the mouse, the chick and the mongoose were still recently used, in particular for preliminary tests before ciguatoxin extraction from fish, since assays in these animals can directly assay the whole flesh. In contrast, various other in vivo methods, such as the kitten, mosquito and diptera larvae assays, were abandoned despite their interesting results. Finally, the mouse neuroblastoma and rat brain synaptosome assays, carried out in vitro as alternative approaches to animal-using assays, are highly sensitive and much more specific than the in vivo methods to detect ciguatoxins.

Erythropoietin Did Not Activate the JAK/STAT or the ERK1/2 Pathway in the Human Colorectal Cell Line, HT-29.

Erythropoietin (EPO) is the cytokine essential for erythropoiesis; however, the expression of EPO and EPO receptor (EPOR) is not limited to cells of erythroid lineage. Erythropoietin and EPOR expression has been localized to numerous non-hematopoietic cells and tissues including endothelial, neuronal and ovarian. Moreover, several tumor types have been shown to express both EPO and EPOR and display increased expression upon hypoxia. To shed light on the potential biological role of EPOR and tumor cells we characterized the response of human colon carcinoma HT-29 cells to EPO. Functionality of the erythropoietin receptor was assessed by radio-labeled ligand binding, cellular proliferation/signaling, and gene expression using DNA microarrays. Receptor binding experiments using [125I]-EPO did not reveal measurable EPO binding activity present on the surface of HT-29 cells under both normoxic and hypoxic conditions. Moreover, EPO failed to induce cellular proliferation or an increase in the phosphorylation state of STAT5, EPOR or ERK1/2 under normoxic or hypoxic conditions at supra-pharmacological levels (25 IU/ml). Gene expression analysis revealed no significant change in gene expression in response to EPO (5 IU/ml) under normoxic conditions. On the other hand, over 347 genes exhibited greater than a 1.5 fold change in gene expression when cells were cultured under hypoxic conditions (1 % O2). When EPO was administered to cells in the hypoxic state, 36 additional genes were observed (9 and 27 up-regulated or down-regulated, respectively). That HT-29 cells exhibit minor transcriptional changes in response to EPO raises the possibility that EPO may signal in HT-29 cells. However, the mechanism for this response is not through the previously described EPO/EPOR signal transduction pathway. This conclusion is supported by the apparent lack of EPO receptor expression on the cell surface. These results suggest that tumor microenvironment, e.g., hypoxia, exerts a greater effect than that seen by exposure to erythropoietin.

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda ( Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/ z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for >90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and <900 Da were suspected to contribute to the overall toxicity.

Differential binding of chemokines to macrophages and neutrophils in the human inflamed synovium.

In chronic inflammatory foci, such as the rheumatoid joint, there is enhanced recruitment of phagocytes from the blood into the tissues. Chemokines are strongly implicated in directing the migration of these cells, although little is known regarding the chemokine receptors that could mediate their chemotaxis into the joint tissue. Therefore the objective of the study was to identify chemokine binding sites on macrophages and neutrophils within the rheumatoid synovium using radiolabeled ligand binding and in situ autoradiography. Specific binding sites for CCL3 (macrophage inflammatory protein-1alpha), CCL5 (RANTES), CCL2 (monocyte chemoattractant protein-1) and CXCL8 (IL-8) were demonstrated on CD68+ macrophages in the subintimal and intimal layers. The number and percentage of intimal cells that bound chemokines were greater in inflamed regions compared to noninflamed regions. The intensity of intimal binding varied between chemokines with the rank order, CCL3 > CCL5 > CCL2 > CXCL8. Neutrophils throughout the synovium bound CXCL8 but did not show any signal for binding CCL2, CCL3 or CCL5. Immunohistochemistry showed that both CXCR1 and CXCR2 are expressed by macrophages and neutrophils in the rheumatoid and nonrheumatoid synovia, suggesting that both of these receptors are responsible for the CXCL8 binding. The chemokine binding sites described on phagocytes may be involved in the migration of these cells into the inflamed joint.

Functional muscarinic M 2 and M 3 receptors and β-adrenoceptor in cultured rat bladder smooth muscle

A highly purified rat urinary bladder smooth muscle cell culture was obtained by a modified enzymic isolation method, and the presence of functional muscarinic as well as β-adrenergic receptors were subsequently determined. At 7–10 days of culture, cells became elongated and spindle-shaped showing a typical “hills and valleys” form. They were stained with anti-α-actin and anti-myosin antibodies. Radiolabeled ligand binding using [ 3H]N-methylscopolamine and [ 3H]CGP12177 showed that these cells expressed muscarinic and β-adrenergic receptors. Stimulation of cultured cells with carbachol inhibited the forskolin-stimulated cyclic AMP formation, caused an elevation of intracellular Ca 2+ concentration measured by fura-2 fluorometry. The latter response was almost completely blocked by 4-DAMP, a selective muscarinic M 3 antagonist. On the other hand, stimulation of cultured cells with isoproterenol enhanced the basal cyclic AMP formation, which was reversed by carbachol. Therefore, the presence of functional muscarinic (both M 2 and M 3) as well as β-adrenergic receptors was confirmed in pure culture of the rat bladder smooth muscle cells obtained by using an enzymic isolation method.

Binding of estrogen and progesterone-BSA conjugates to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the effects of the free steroids on GAPDH enzyme activity: physiological implications

In this study rat brain solubilized plasmalemma-microsomal fractions (B-P3) or cytosolic fractions were applied to P-3-BSA (progesterone linked to BSA at C-3 position) and E-6-BSA (17β-estradiol linked to BSA at C-6 position) affinity columns. It is interesting that a 37 kDa protein was retained by both columns which was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by N-terminal sequencing. The 37 kDa protein (GAPDH) was not retained by either a control BSA conjugated affinity column or a corticosterone-BSA affinity column. E-6-BSA bound to GAPDH with higher binding affinity than P-3-BSA or T-3-BSA (testosterone linked to BSA at C-3 position) affinity columns. In addition, the binding of 17β-E-6-BSA to GAPDH was impeded by free estrogen (17β-estradiol) completely. Binding studies of E-6-BSA and P-3-BSA to commercial GAPDH from rabbit skeletal muscle using radiolabeled ligand binding assays revealed that P-3-BSA had 10× lower GAPDH binding affinity than E-6-BSA. Next, the effects of estrogen and progesterone on GAPDH activity were studied. Rapid and significant increases in V max and changes in K m were observed by the addition of 10 nM estradiol, whereas 100 nM progesterone decreased only V max significantly. Testosterone, corticosterone, 17α-estradiol, and diethylstilbestrol did not affect the enzyme activity. The results indicate that GAPDH is a target site for 17β-estradiol and progesterone and suggest possible roles in the regulation of cellular metabolism and synaptic remodeling in which GAPDH has been reported to be involved.

Upregulation of V(1) receptors in renal resistance vessels of rats developing genetic hypertension.

Previous studies have demonstrated that arginine vasopressin (AVP) produces exaggerated renal vasoconstriction in young spontaneously hypertensive rats (SHR) relative to normotensive rats. The exaggerated renal vascular reactivity does not appear to be due to a primary defect in postreceptor calcium signal transduction. Although the magnitudes of vascular responses differ, the relative proportions of calcium entry and mobilization pathways evoked by AVP in renal resistance vessels are similar in these rat strains. The purpose of the present study was to evaluate possible differences in V(1) mRNA and receptor density and affinity in preglomerular resistance vessels (<50 microm) obtained from young Wistar-Kyoto (WKY) and SHR. Quantitative RT-PCR analysis revealed twofold greater expression of the V(1a) receptor gene in preglomerular arterioles of 7-wk-old SHR compared with WKY. In vitro radiolabeled ligand binding studies were performed under equilibrium conditions on preglomerular resistance arterioles freshly isolated from kidneys of 7-wk-old rats. The results indicate that AVP receptor density (B(max)) is two to three times greater in SHR than in WKY (248 +/- 24 vs. 91 +/- 11 fmol/mg protein, P < 0.001). The affinity does not differ between strains (K(d) = 0.5 nM). Displacement studies yielded similar results for SHR and WKY. Unlabeled AVP completely displaced [(3)H]AVP binding, with an IC(50) of 2.5 x 10(-10) M. Expression of AVP receptor types in afferent arterioles was evaluated using the V(1) receptor agonist, [Phe(2), Ile(3),Org(8)]vasopressin, the V(1) receptor antagonist, [d(CH(2))(5), Tyr(Me)(2), Tyr(NH(2))(9)]Arg(8)-vasopressin, and the V(2) receptor agonist, desamino-[D-Arg(8)]vasopressin. Both the V(1) agonist and antagonist displaced up to 90% of the AVP binding with IC(50) values of 4 x 10(-8) and 8 x 10(-7) M, respectively. The V(2) receptor agonist was a weak inhibitor, displacing less than 15% of AVP binding at a high concentration of 10(-4) M. These results demonstrate that virtually all AVP receptors in the preglomerular arterioles are of the V(1) type. Collectively, our results provide evidence that the enhanced renal reactivity to AVP is mediated by a higher density of V(1) receptors associated with increased gene expression in renal resistance vessels of SHR developing genetic hypertension.

Cutting edge: identification of a novel chemokine receptor that binds dendritic cell- and T cell-active chemokines including ELC, SLC, and TECK.

Searching for new receptors of dendritic cell- and T cell-active chemokines, we used a combination of techniques to interrogate orphan chemokine receptors. We report here on human CCX CKR, previously represented only by noncontiguous expressed sequence tags homologous to bovine PPR1, a putative gustatory receptor. We employed a two-tiered process of ligand assignment, where immobilized chemokines constructed on stalks (stalkokines) were used as bait for adhesion of cells expressing CCX CKR. These cells adhered to stalkokines representing ELC, a chemokine previously thought to bind only CCR7. Adhesion was abolished in the presence of soluble ELC, SLC (CCR7 ligands), and TECK (a CCR9 ligand). Complete ligand profiles were further determined by radiolabeled ligand binding and competition with >80 chemokines. ELC, SLC, and TECK comprised high affinity ligands (IC50 <15 nM); lower affinity ligands include BLC and vMIP-II (IC50 <150 nM). With its high affinity for CC chemokines and homology to CC receptors, we provisionally designate this new receptor CCR10.

The proliferative response to platelet-derived growth factor of smooth muscle cells isolated from synthetic vascular grafts in a canine model

Objective: Previous studies on graft healing have shown increased platelet-derived growth factor (PDGF) production in graft segments versus native aortic segments. The purpose of this study was to characterize the proliferative response of graft smooth muscle cells (SMCs) to PDGF. Methods: Thoracoabdominal grafts were implanted in beagles. SMCs were harvested from the graft and the proximal and distal aortas. Basal proliferation was assessed with growth curves in primary culture. The proliferative response to PDGF then was compared with [ 3H]thymidine uptake studies and cell counts. Finally, PDGF receptors were characterized with radio-labeled ligand binding assays. Results: The growth curves showed that the graft SMCs entered log-phase growth 2 days earlier than did the aortic SMCs. Stimulation of quiescent early-passage graft SMCs with PDGF (10 ng/mL) resulted in a 1.7 ± 0.1–fold increase in [ 3H]thymidine incorporation, which was significantly less than that of the SMCs from both the proximal aorta (11.8 ± 3.0) and the distal aorta (10.2 ± 1.9; P < .5). Similarly, the 1.1 ± 0.1–fold increase in graft SMC cell number was significantly less than the increases for both proximal (2.8 ± 0.5) and distal (2.9 ± 0.8) aortic SMCs ( P < .5). Binding studies on quiescent first-passage cells showed fewer PDGF receptors available for binding in the graft SMCs (185 ± 70 fmol/million cells) as compared with both the proximal (419 ± 147 fmol/million cells) and the distal (387 ± 112 fmol/million cells) aortas ( P < .5). Binding affinity was similar for the three groups. Conclusion: Graft SMCs exist in a chronic proliferative state but exhibit a decreased proliferative response to PDGF and have fewer receptors available for binding PDGF than do aortic SMCs in vitro. (J Vasc Surg 1999;29:845-51.)

De novo design, synthesis, and biological activities of high-affinity and selective non-peptide agonists of the delta-opioid receptor.

On the basis of the structure-activity relationships of delta-opioid-selective peptide ligands and on a model of the proposed bioactive conformation for a potent and selective, conformationally constrained delta-opioid peptide ligand [(2S, 3R)-TMT1]DPDPE, a series of small organic peptide mimetic compounds targeted for the delta-opioid receptor have been designed, synthesized, and evaluated in radiolabeled ligand binding assays and in vitro bioassays. The new non-peptide ligands use piperazine as a template to present the most important pharmacophore groups, including phenol and phenyl groups and a hydrophobic moiety. This hydrophobic group was designed to mimic the hydrophobic character of the D-Pen residues in DPDPE, which has been found to be extremely important for increasing the binding affinity and selectivity of these non-peptide ligands for the delta-opioid receptor over the mu-opioid receptor. Compound 6f (SL-3111) showed 8 nM binding affinity and over 2000-fold selectivity for the delta-opioid receptor over the mu-opioid receptor. Both enantiomers of SL-3111 were separated, and the (-)-isomer was shown to be the compound with the highest affinity for the delta-opioid receptor found in our study (IC50 = 4.1 nM), with a selectivity very similar to that observed for the racemic compound. The phenol hydroxyl group of SL-3111 turned out to be essential to maintain high affinity for the delta-opioid receptor, which also was observed in the case of the delta-opioid-selective peptide ligand DPDPE. Binding studies of SL-3111 and [p-ClPhe4]DPDPE on the cloned wild-type and mutated human delta-opioid receptors suggested that the new non-peptide ligand has a binding profile similar to that of DPDPE but different from that of (+)-4-[((alphaR)-alpha(2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80), another delta-opioid-selective non-peptide ligand.

Characterization of the High Affinity Cell-binding Domain in the Cell Surface Proteoglycan Syndecan-4

The syndecan family of cell surface proteoglycans regulates cell adhesion via their glycosaminoglycan chains and discrete domains of their core proteins. Core protein domains that are variable between syndecan family members may regulate syndecan-specific associations, thereby endowing individual syndecans with unique functions. A syndecan-4-specific domain has been identified in the extracellular syndecan-4 protein. This region mediates cell adhesion when provided as an artificial substratum and is localized within amino acids 56-109 of the recombinant extracellular protein domain of mouse syndecan-4 (mS4ED) (McFall, A. J., and Rapraeger, A. C. (1997) J. Biol. Chem. 272, 12901-12904). To characterize its interaction with the cell surface, radiolabeled ligand binding studies were performed. A single high affinity interaction, with a dissociation constant of 2 x 10(-9) M, was observed between mS4ED and both human and mouse cells. Both chicken S4ED and mS4ED compete for this interaction, although they are only 34% identical within the cell-binding domain sequence. The extracellular protein domains of syndecan-1, -2, and -3, however, fail to compete. The interaction is also observed with native syndecan-4 shed from cell surfaces. Interestingly, the extracellular protein domain of syndecan-1 also mediates cell adhesion, suggesting a similar but discrete interaction for this family member.