Half-maximal Displacement Research Articles

The rate of dasotraline brain entry is slow following intravenous administration

RationaleDrugs that rapidly increase dopamine levels have an increased risk of abuse. Dasotraline (DAS) is a dopamine and norepinephrine reuptake inhibitor characterized by slow oral absorption with low potential for abuse. However, it remains unclear whether intravenous (i.v.) administration would facilitate the rapid elevation of dopamine levels associated with stimulant drugs.ObjectiveTo assess the kinetics of DAS across the blood-brain barrier and time to onset of dopamine transporters (DAT) inhibition.MethodsWe compared the onset of DAT occupancy and the associated elevation of synaptic dopamine levels in rhesus monkey following i.v. administration of DAS or methylphenidate (MPH) using positron emission tomography (PET). Brain entry times were estimated by reductions in [18F]-FE-PE2I binding to DAT in rhesus monkeys. Elevations of synaptic dopamine were estimated by reductions in [11C]-Raclopride binding to D2 receptors.ResultsIntravenous administration of DAS (0.1 and 0.2 mg/kg) resulted in striatal DAT occupancies of 54% and 68%, respectively; i.v. administered MPH (0.1 and 0.5 mg/kg) achieved occupancies of 69% and 88% respectively. Brain entry times of DAS (22 and 15 min, respectively) were longer than for MPH (3 and 2 min). Elevations in synaptic dopamine were similar for both DAS and MPH however the time for half-maximal displacement by MPH (t = 23 min) was 4-fold more rapid than for DAS (t = 88 min).ConclusionsThese results demonstrate that the pharmacodynamics effects of DAS on DAT occupancy and synaptic dopamine levels are more gradual in onset than those of MPH even with i.v. administration that is favored by recreational drug abusers.

Antigenicity and Epitope Specificity of ZnT8 Autoantibodies in Type 1 Diabetes

The single nucleotide polymorphism (SNP) rs13266634 encodes either an Arginine (R) or a Tryptophan (W) (R325W) at the amino acid position 325 in the Zinc Transporter 8 (ZnT8) protein. Autoantibodies (Ab) that recognize ZnT8R, ZnT8W or both at the polymorphic site are common in newly diagnosed type 1 diabetes (T1D) patients. The epitope specificity and affinity of ZnT8Ab are poorly understood, but may be of importance for the prediction and clinical classification of T1D. Therefore, the aims were to 1) determine the immunogenicity of short (318-331) ZnT8 peptides in mice and 2) test the affinity of short and long (268-369) ZnT8proteins in T1D patients positive for either ZnT8RAb or ZnT8wAb. Sera from BALB/cByJ mice immunized with short R, W or Q (Glutamine) ZnT8 peptides were tested for ZnT8-peptide antibodies in ELISA and radiobinding assay (RBA). Using reciprocal permutation experiment, short synthetic ZnT8R and ZnT8W (318-331) and long in vitro transcription translation ZnT8R and ZnT8W (268-369) proteins were tested in competitive RBA with R- and W-monospecific T1D sera samples. All mouse sera developed non-epitope-specific peptide antibodies in ELISA and only 6/12 mice had ZnT8-RWQ antibodies in RBA. Both long ZnT8R and ZnT8W (268-369), but not any short, proteins displaced labelled ZnT8 (268-369) proteins in binding to T1D ZnT8Ab-specific sera. The reciprocal cross-over tests showed that half-maximal displacement varied 2- to 11-fold indicating variable affinity of patient ZnT8Ab, signifying crucial autoantibody epitope spreading. The present approach should make it possible to dissect the importance of the R325W ZnT8 autoantigen epitope in the T1D pathogenesis.

Caffeine Occupancy of Human Cerebral A1 Adenosine Receptors: In Vivo Quantification with 18F-CPFPX and PET

Caffeine is the neuroactive agent in coffee and tea and is a broadly consumed stimulant. It is a nonselective antagonist of the neuromodulator adenosine and, if applied in commonly consumed doses, evokes its stimulating effects through the blockade of adenosine receptors. (18)F-8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ((18)F-CPFPX) has been established as a highly selective and affine PET ligand for the A(1) adenosine receptor (A(1)AR). The objective of the present study was to visualize and quantify the in vivo occupancy of the human cerebral A(1)AR by caffeine using (18)F-CPFPX and PET. Fifteen subjects (age range, 24-68 y) underwent a 140-min bolus-plus-constant-infusion PET experiment after at least 36 h of caffeine abstinence. Metabolite-corrected blood data were used to calculate steady-state distribution volumes (V(T)) during the baseline condition of the scan between 70 and 90 min. Subsequently, subjects received a 10-min infusion of varying concentrations (0.5-4.3 mg/kg of body weight) of caffeine at 90 min. Occupancy V(T) of the A(1)AR was thereafter estimated using data acquired between 120 and 140 min. Occupancy levels were calculated using the Lassen plot, from which the inhibitory concentrations of 50% were derived. Plasma levels of caffeine were determined at regular intervals. One subject received an intravenous vehicle as a placebo. Caffeine displaced 5%-44% of (18)F-CPFPX binding in a concentration-dependent manner. There was no change of radioligand binding after the administration of placebo. Half-maximal displacement was achieved at a plasma caffeine concentration of 67 μM, which corresponds to 450 mg in a 70-kg subject or approximately 4.5 cups of coffee. Given a biologic half-life of about 5 h, caffeine might therefore occupy up to 50% of the cerebral A(1)AR when caffeinated beverages are repeatedly consumed during a day. Furthermore, the present study provides evidence that (18)F-CPFPX PET is suitable for studying the cerebral actions of caffeine, the most popular neurostimulant worldwide.

A novel cardiac magnetic resonance imaging technique to evaluate left ventricular diastolic function in patients with hypertrophic cardiomyopathy

Background The temporal and spatial resolution of cardiac magnetic resonance (CMR) imaging allows precise tracking of the left ventricle (LV) atrioventricular junction (AVJ) throughout the cardiac cycle. Similar to tissue Doppler imaging in echocardiography, AVJ motion assessed by CMR reflects diastolic LV function. A prior study by our group demonstrated significant differences in the displacement and velocity of the AVJ during diastole in patients with heart failure with preserved ejection fraction compared to normal hearts. In the current study we hypothesized that diastolic left ventricular function defined by AVJ motion is abnormal in patients with hypertrophic cardiomyopathy (HCM), a group known to have altered LV relaxation properties. Methods We retrospectively identified 24 patients with known or suspected HCM and normal ejection fractions who underwent CMR as part of their clinical assessment and 14 normal control subjects. The longitudinal motion of the lateral and septal AVJs was tracked at 25 times through the cardiac cycle using the 4-chamber cine CMR view . The baseline position of the AVJ was defined at end diastole and its longitudinal displacement was measured relative to a reference line drawn between the LV apex and the midpoint of the mitral annnulus. The displacement of the AVJ in relation to its starting position at end diastole was normalized by LV length. Based on the resulting plots of AVJ position versus time in the cardiac cycle (see figure below) five motion variables were calculated: maximum longitudinal displacement (MD) of the AVJ, maximum velocity during early diastole (MVED), velocity at half-maximal displacement during early diastole (VHMDED), slope of the best fit line of displacement in diastasis (VDS), and the ratio of VDS/MVED. Data obtained in HCM patients were compared to normal control subjects using the Student t-test. Results Means and standard deviations were calculated for each of the AVJ motion variables (MD, VHMDED, MVED, VDS, VDS/MVED) for the septal (S) and lateral walls (LW) of HCM patients and normal control subjects. Lateral and septal AVJ data were directly compared to the corresponding walls in patients and controls. We found highly statistically significant differences in MD and the four CMR correlates of diastolic LV function (VHMDED, MVED, VDS, VDS/MVED) in patients with HCM compared to normal controls, as noted in the table below. Conclusions Novel CMR correlates of diastolic left ventricular function are markedly abnormal in patients with HCM. Measured at the AVJ, patients with HCM had significantly less maximal displacements, slower velocities during early diastolic filling, and higher velocities during diastasis compared to normal control subjects. The evaluation of AVJ motion by CMR may provide a new method to assess diastolic function.

The Silent K+ Channel Subunit, KV6.4. Influences the Gating Charge Movement of KV2.1 in a Heterotetrameric Channel Complex

Voltage-gated K+ (Kv) channels are tetramers of α-subunits that detect changes in membrane potential (V) by a positively charged (Q) voltage-sensing domain (VSD). Molecular movements of VSDs lead to charge displacement that can be recorded as transient gating currents (IQ), which subsequently results in channel gating. The silent Kv subunit, Kv6.4, does not form functional homotetramers; however, it can tetramerize with Kv2.1 subunits to form functional Kv2.1/Kv6.4 heterotetramers, with a proposed 3:1 stoichiometry. Previously we showed that Kv6.4 subunits exert a significant (∼40 mV) hyperpolarizing shift in the voltage-dependent inactivation of heterotetrameric Kv2.1/Kv6.4 channels, as compared to Kv2.1 homotetramers, without significant effects on activation gating. However, the underlying mechanism remains unclear. To address this we analyzed ionic and IQ recordings from heterotetrameric Kv2.1/Kv6.4 channels transiently expressed in HEK293A cells. Half-maximal displacement of gating charge (Q1/2) for Kv2.1 homotetramers was −26 mV, as determined from the charge-voltage (Q-V) curve. Analysis of the decay time constant of IQ-ON as a function of voltage resulted in a bell shaped curve with a maximal time constant around the midpoint potential of −20 mV. Co-expressing Kv6.4 with Kv2.1 resulted in earlier charge movement as evident from a ∼16 mV hyperpolarizing shift in the Q-V curve. Furthermore, we observed a double bell shaped curve for the decay time constant, with maximal time constants around −20 mV and −70 mV; the latter corresponding to the Kv6.4-induced ∼40 mV hyperpolarizing shift in the voltage-dependence of channel inactivation. Therefore, we suggest that this more negatively located ON-gating component presumably reflects the voltage-dependence of the Kv6.4 subunit within the Kv2.1/Kv6.4 heterotetramer, and that the VSD movement of only the Kv6.4 subunit is sufficient to induce closed state channel inactivation.

Crystal Structure of Lactadherin C2 Domain at 1.7Å Resolution with Mutational and Computational Analyses of Its Membrane-binding Motif

Lactadherin is a phosphatidyl-L-serine (Ptd-L-Ser)-binding protein that decorates membranes of milk fat globules. The major Ptd-l-Ser binding function of lactadherin has been localized to its C2 domain, which shares homology with the C2 domains of blood coagulation factor VIII and factor V. Correlating with this homology, purified lactadherin competes efficiently with factors VIII and V for Ptd-L-Ser binding sites, functioning as a potent anticoagulant. We have determined the crystal structure of the lactadherin C2 domain (Lact-C2) at 1.7A resolution. The bovine Lact-C2 structure has a beta-barrel core that is homologous with the factor VIII C2 (fVIII-C2) and factor V C2 (fV-C2) domains. Two loops at the end of the beta-barrel, designated spikes 1 and 3, display four water-exposed hydrophobic amino acids, reminiscent of the membrane-interactive residues of fVIII-C2 and fV-C2. In contrast to the corresponding loops in fVIII-C2 and fV-C2, spike 1 of Lact-C2 adopts a hairpin turn in which the 7-residue loop is stabilized by internal hydrogen bonds. Further, central glycine residues in two membrane-interactive loops may enhance conformability of Lact-C2 to membrane binding sites. Mutagenesis studies confirmed a membrane-interactive role for the hydrophobic and/or Gly residues of both spike 1 and spike 3. Substitution of spike 1 of fVIII-C2 into Lact-C2 also diminished binding. Computational ligand docking studies identified two prospective Ptd-l-Ser interaction sites. These results identify two membrane-interactive loops of Lact-C2 and provide a structural basis for the more efficient phospholipid binding of lactadherin as compared with factor VIII and factor V.

Activation mechanisms for the cystic fibrosis transmembrane conductance regulator protein involve direct binding of cAMP.

The CFTR [CF (cystic fibrosis) transmembrane conductance regulator] chloride channel is activated by cyclic nucleotide-dependent phosphorylation and ATP binding, but also by non-phosphorylation-dependent mechanisms. Other CFTR functions such as regulation of exocytotic protein secretion are also activated by cyclic nucleotide elevating agents. A soluble protein comprising the first NBD (nucleotide-binding domain) and R-domain of CFTR (NBD1-R) was synthesized to determine directly whether CFTR binds cAMP. An equilibrium radioligand-binding assay was developed, firstly to show that, as for full-length CFTR, the NBD1-R protein bound ATP. Half-maximal displacement of [3H]ATP by non-radioactive ATP at 3.5 microM and 3.1 mM was demonstrated. [3H]cAMP bound to the protein with different affinities from ATP (half-maximal displacement by cAMP at 2.6 and 167 microM). Introduction of a mutation (T421A) in a motif predicted to be important for cyclic nucleotide binding decreased the higher affinity binding of cAMP to 9.2 microM. The anti-CFTR antibody (MPNB) that inhibits CFTR-mediated protein secretion also inhibited cAMP binding. Thus binding of cAMP to CFTR is consistent with a role in activation of protein secretion, a process defective in CF gland cells. Furthermore, the binding site may be important in the mechanism by which drugs activate mutant CFTR and correct defective DeltaF508-CFTR trafficking.

Crystal Structure of the Bovine Lactadherin C2 Domain, a Potential Anticoagulant, Shows Similarity to Factor V and Factor VIII.

Lactadherin is a glycoprotein expressed by mammary epithelial cells as a cell surface protein and as an abundantly secreted protein during lactation. Lactadherin is comprised of two EGF-like domains and two C-like domains that share homology with the C domains of blood clotting proteins Factor V (FV) and Factor VIII (FVIII). Similar to these coagulation factors, lactadherin binds to phosphatidylserine (PS) containing phospholipid (PL) membranes with high affinity although lactadherin binding requires lower concentrations of PS and is independent of phosphatidylethanolamine. Lactadherin shows efficient competition for membrane binding sites recognized by vitamin K-dependent coagulation factors as well as FV and FVIII with half-maximal displacement of these proteins from PS containing PL vesicles at lactadherin concentrations of 1–4 nM. Lactadherin inhibits the tenase complex, the prothrombinase complex, and the factor VIIa-tissue factor complex. Thus, it has been suggested as an anticoagulant through competition for PL binding sites with blood coagulation proteins. As blood coagulation factors VIII and V bind to PL membranes via C domains with high affinity and specificity, we have determined the crystal structure of the C2 domain of bovine lactadherin (residues 1 to 158) for comparison with the crystal structures of the C2 domains of FV and FVIII. The lactadherin C2 domain was cloned from a bovine EST clone, over-expressed in Pichia pastoris, purified by ion-exchange and gel filtration chromatography and crystallized by the vapor diffusion method. The crystals diffracted to 2.4 Å and have cell dimension of a=108.12Å, b=107.79Å, c= 82.75Å and belong to space group P212121. The structure was determined and refined at a resolution of 2.4 Å. There are four molecules per asymmetry unit. The overall structure of the lactadherin C2 is similar to the C2 domains of FV and FVIII (root-mean-square-deviation of Cα atoms of 0.9 Å and 1.2 Å, respectively). Similarly to the FV (Macedo-Ribeiro, S et al, Nature 1999, 402:434–439) and FVIII (Pratt, KP et al, Nature, 1999, 402:439–441) C2 domains, the lactadherin C2 domain consists of eight major antiparallel strands arranged in two β-sheets of five and three strands packed against one another with the N- and C-terminal regions linked by a disulfide bridge. Like the FV and FVIII C2 domains, the lactadherin C2 domain structure reveals a β-sheet-sandwiched core, from which two β-turns and a loop display a group of solvent-exposed hydrophobic residues. The crystal structures suggest that PL binding is mediated by hydrophobic residues in the C2 domains of FV and FVIII and multiple mutagenesis and antibody inhibition studies support this hypothesis. Based on the crystal structure it is likely that residues Trp26, Phe31, Thr53, Phe81, and Gly82 of lactadherin participate in PL binding. The conformations of regions involved in PL binding are quite different from that of FV and FVIII. The C2 domain of lactadherin may thus have the potential to serve as a unique anticoagulant for treatment of thrombotic disease by blocking the action of FV and FVIII.

Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites

Lactadherin, a glycoprotein of the milk-fat globule membrane, contains tandem C domains with homology to discoidin-type lectins and to membrane-binding domains of blood-clotting factors V and VIII. We asked whether the structural homology confers the capacity to compete for the membrane-binding sites of factor VIII and factor V and to function as an anticoagulant. Our results indicate that lactadherin competes efficiently with factor VIII and factor V for binding sites on synthetic phosphatidylserine-containing membranes with half-maximal displacement at lactadherin concentrations of 1 to 4 nM. Binding competition correlated to functional inhibition of factor VIIIa-factor IXa (factor Xase) enzyme complex. In contrast to annexin V, lactadherin was an efficient inhibitor of the prothrombinase and the factor Xase complexes regardless of the degree of membrane curvature and the phosphatidylserine content. Lactadherin also inhibited the factor VIIa-tissue factor complex efficiently whereas annexin V was less effective. Because the inhibitory concentration of lactadherin was proportional to the phospholipid concentration, and because lactadherin was not an efficient inhibitor in the absence of phospholipid, the major inhibitory effect of lactadherin relates to blocking phospholipid sites rather than forming inhibitory protein-protein complexes. Lactadherin was also an effective inhibitor of a modified whole blood prothrombin time assay in which clotting was initiated by dilute tissue factor; 60 nM lactadherin prolonged the prothrombin time 150% versus 20% for 60 nM annexin V. These results indicate that lactadherin can function as a potent phospholipid-blocking anticoagulant.

Purification and characterization of a novel intracellular 17 beta-estradiol binding protein in estrogen-resistant New World primate cells.

Compared to Old World primates, including man, New World primates display target-tissue resistance to gonadal steroid hormones. In female New World primates this resistant phenotype is characterized by elevated concentrations of plasma estradiol and progesterone. Here we describe the discovery of an intracellular estrogen binding protein (IEBP) that acts to concentrate 17 beta-estradiol (E2) in the cytoplasm of New World primate target cells. IEBP was purified by E2-affinity chromatography from postnuclear extracts of the B95-8 cells established from an E2-resistant New World primate. Compared with unpurified extract, affinity-purified IEBP demonstrated a 300-fold enrichment in specific E2 binding activity; half-maximal displacement of [3H]E2 from affinity-purified IEBP was observed with 0.1 nM E2. Affinity-purified extracts were subjected to SDS-PAGE with isolation of a dominant 27-28 kDa protein. N-terminal sequencing of tryptic peptides of the protein showed sequence homology with human heat shock protein-27 (hsp27). By immunoblot and E2 binding capacity, IEBP was 1] 2-3-fold greater in New World than in Old World primate tissues and cell lines, 2] heat-inducible and 3] up-regulated in vivo in the presence of the functioning female gonad. In conclusion, IEBP is a specific E2-interacting heat shock protein in the hsp-27 family that is relatively overexpressed in estrogen-resistant cells.

Lipoxin A4: a new class of ligand for the Ah receptor.

The Ah receptor is a ligand-activated transcription factor that mediates many of the biological actions of a large class of environmental compounds. Support for a role of the Ah receptor in normal physiology also has been reported, but an endogenous regulating ligand has not been identified. We have examined candidate endogenous lipophilic substances and report here the ability of the arachidonic acid metabolite, lipoxin A4, to bind to and activate the Ah receptor in Hepa-1 cells. Lipoxin A4 produced a concentration-dependent response in a DRE-driven CAT reporter construct, with a greater than 10-fold increase in CAT activity at 0. 3 microM. Lipoxin A4 transformed the Ah receptor to an active DRE-binding form in a concentration-dependent manner as indicated by gel mobility shift analysis. Results of Ah receptor competitive binding experiments indicated that at a concentration of 100 nM, lipoxin A4 produced a half-maximum displacement (EC50) of [3H]TCDD binding. Results of Northern blot analyses indicated a transient increase in mRNA levels of the Ah receptor-responsive gene CYP1A1, which peaked at 4 h, consistent with the kinetics observed for lipoxin A4-induced CYP1A1 enzyme activity. Further, lipoxin A4 was found to be a competitive inhibitor for the CYP1A1 enzyme, with a calculated Ki = 1.1 microM. These results establish lipoxin A4 as a new class of Ah receptor ligand, one that differs dramatically from classical Ah receptor ligands.

Calcitonin gene-related peptide receptor in human placental syncytiotrophoblast brush-border and basal plasma membranes

Minerals, such as calcium and potassium, are essential for fetal development, but their transplacental transport, and in particular, the effect of hormones on this process has not been extensively studied. Human α-calcitonin gene-related peptide (hαCGRP), a hormone constituted of 37 amino acids, is obtained by the alternative splicing of the mRNA from the calcitonin gene, and could be implicated in placental ion transport. In order to study the presence of this receptor, brush-border and basal plasma membranes were purified, and membrane binding studies were conducted using [ 125I]hαCGRP. The initiation of binding of [ 125I]hαCGRP to both membranes was rapid and reached maximal value after 10 min of incubation at 37°C. Scratchard analysis revealed single-affinity binding sites for hαCGRP with K d equal to 4412.45 ± 604.81 p m and 2673.24 ± 552.51 p m for brush-border and basal plasma membranes, respectively, which were significantly different. Moreover, the maximal number of receptors was significantly different ( P<0.001) in both membranes, with B max of 627.94 ± 31.40 fmol/mg protein for brush-border membranes and 343.70 ± 43.52 fmol/mg protein in basal-plasma membranes. Competitive displacement of [ 125I]hαCGRP with other ligands showed the following potencies; hαCGRP ≈ hβCGRP ≈ Cys (acm) 2,7 CGRP>CGRP (8–37), but no competition was observed with human and salmon calcitonin. Half-maximal displacement for human α- and βCGRP was reached at approximately 10 −10 m for brush-border and basal-plasma membranes. α- and βCGRP, and their fragment and analogue, stimulated cyclic AMP production in placental homogenate ranging from 143–163 per cent. Thus, our results show the presence of CGRP-specific receptors in both the syncytiotrophoblast membranes of human placenta. The role(s) of this related peptide in placenta remains to be investigated

Different binding and degradation of proinsulin, insulin and insulin-like growth factor-1 (IGF-1) in cultured renal proximal tubular cells. Implications for the prolonged serum half-life of proinsulin.

Recent studies have reported that elevated proinsulin levels are indicative of an increased cardiovascular risk. Renal proximal tubular cells represent a major site for the metabolism of insulin-like hormones after glomerular filtration into the tubular lumen. To determine the binding and degradation of proinsulin in comparison with insulin and insulin-like growth factor-1 (IGF-1), we have used a rabbit proximal tubular cell line (PT-1). As confirmed by electron microscopy. PT-1 cells exhibit bipolar differentiation, demonstrating apical microvilli and invaginations of the basolateral membrane. To allow selective incubation of both compartments, cells were grown on filter membranes. Performing equilibrium binding assays with 125I-labelled hormones, severalfold higher binding was found at the apical than at the basolateral cell membrane, with the capacity range IGF-1 > insulin > proinsulin. Half-maximal displacement of 125I-labelled insulin and IGF-1 was observed at 0.6 and 2 nM, respectively, while crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Half-maximal displacement of 125I-proinsulin binding was obtained at approx. 8 nM proinsulin and insulin, whereas IGF-1 was 10-fold less potent. The relative degradation of specifically bound tracer was lowest for proinsulin (apical 10%, basolateral: 13%). IGF-1 was degraded by 20% at the apical cell membrane, and up to 78% at the basolateral membrane. In contrast, almost the total amount of insulin bound was degraded at both membrane sites (apical 99%, basolateral: 83%). These results suggest separate insulin and IGF-1 receptors while proinsulin binds with high affinity to a third insulin-like receptor on the apical membrane of PT-1 cells.

Characterization of receptors for insulin-like growth factor type I on cultured human endometrial stromal cells: downregulation by progesterone.

Because insulin-like growth factor type I (IGF-I) is reputed to be involved in the endometrial decidualization, we analyzed the expression of IGF-I receptors in an in vitro system of human endometrial stromal cells. Competitive binding studies of both intact stromal cells and membrane preparation indicated the presence of specific components with high affinity for binding IGF-I. Half-maximum displacement was obtained with 2.3 nmol/l native IGF-I, whereas insulin was unable to achieve half-maximum displacement even at higher concentrations. This IGF-I binding component was found to be a saturable protein in respect of the radioligand [125I]IGF-I, with a dissociation constant of 0.16 nmol/l. Affinity cross-linking studies revealed a labelled band of approximate relative molecular mass 135000, corresponding to the known alpha-subunit of IGF-I receptor. This band was significantly inhibited dose-dependently by the IGF-I receptor monoclonal antibody alpha-IR3 or native IGF-I, suggesting that the IGF-I binding component in the membrane of stromal cells has the identity of the alpha-subunit of IGF-I receptor. Cell proliferation in vitro was stimulated by progesterone. Furthermore, progesterone downregulated the [125I]IGF-I binding activity by downregulation of the IGF-I membrane receptor of human endometrial stromal cells. These data show that the IGF-I receptor is a functionally integral component of the stromal cell membrane structure, and its expression might be directly modulated by progesterone and, therefore, might play an important role in the preparation of the stroma for successful embryo implantation.

Particulate binding sites for steroid hormones in subcellular fractions of the ovine corpus luteum: properties and hormone specificity

We have described previously the presence of binding sites in particulate fractions of the porcine corpus luteum (CL) which were specific for progesterone. We now demonstrate the presence of similar progesterone-specific binding sites in particulate fractions of the ovine CL. Preincubation of ovine luteal membranes with radiolabelled steroids demonstrated binding of progesterone and pregnenolone to a low-density particulate fraction (1.07–1.09 g/cm 3). Preincubation with digitonin perturbed the buoyant density of this fraction (to1.10–1.14 g/cm 3) without causing release of steroid. Androgens and oestrogens did not bind appreciably to control luteal membranes, but were bound when preincubated with digitonin. In contrast, steroid conjugates (oestrone sulfate, pregnanediol glucuronide), cortisol, fatty acids (arachidonic acid, prostaglandin F 2α) and cholesterol ester failed to bind to ovine luteal membranes, with or without digitonin pretreatment. The effects of digitonin on steroid binding led us to examine its effects on steroid binding to ovine luteal membrane fractions in vitro. Specific progesterone binding was absent in the absence of digitonin, even at very high membrane concentrations. However, binding of 3H-labelled progesterone was stimulated 5–15-fold in a dose-dependent fashion by increasing digitonin concentrations, reaching a plateau at about 100 μM. In the presence of digitonin, [ 3H]progesterone binding increased linearly with luteal membrane concentration. Other detergents, saponins and cardiotonic steroids tested did not stimulate progesterone binding to ovine luteal membranes. [ 3H]Progesterone binding was dependent on the pH, duration and temperature of incubation. Unlabelled progesterone decreased binding of [ 3H]progesterone (half-maximal displacement of specific binding (IC 50) at about 60 nM) whereas androgens were less potent (IC 50, 500–3300 nM), whilst a wide range of other steroids and inhibitors of steroidogenic enzymes were neffective, except at very high concentrations. Similarly, a number of progesterone receptor agonist and antagonist analogues failed to compete for progesterone binding to luteal membranes, suggesting that these binding sites were unrelated to progesterone receptors. Modifications to the 3,4, 5 and 11 positions of progesterone, removal of the steroid side-chain or aromatization of the A-ring decreased binding potency dramatically, whereas changes to the 17 or 20 positions had relatively minor effects. Our results indicate the presence of a low density particulate fraction in ovine corpora lutea which contains specific binding sites for endogenous and exogenous progesterone.

Differential interaction of glimepiride and glibenclamide with the β-cell sulfonylurea receptor I. Binding characteristics

Glimepiride is a novel sulfonylurea drug for treatment of non-insulin-dependent diabetes mellitus with higher blood sugar lowering efficacy in diabetic patients than glibenclamide raising the question whether this characteristics is in line with different binding of glimepiride and glibenclamide to the β-cell sulfonylurea receptor. Scatchard plot analysis of [ 3H]sulfonylurea binding to membranes isolated from rat β-cell tumors and (RINm5F) insulinoma cells and to RINm5F cells demonstrated that glimepiride has a 2.5–3-fold lower affinity than glibenclamide. This corresponded well to the 8–9-fold higher k off and 2.5–3-fold higher k on rates of glimepiride compared to glibenclamide as revealed by the dissociation and association kinetics of [ 3H]sulfonylurea binding and the K d values calculated thereof. In agreement, the concentrations required for half-maximal displacement of [ 3H]sulfonylurea bound to β-cell membranes were significantly higher for glimepiride compared to glibenclamide. However, the binding affinity of glimepiride measured by both equilibrium binding and kinetic binding studies upon solubilization of β-cell tumor membranes and RINm5F cell membranes increased up to the value for glibenclamide. This was primarily based on a drastic decrease of the dissociation rate constant of glimepiride whereas the kinetics of glibenclamide binding remained largely unaffected upon solubilization. These data suggest that the K d value alone is not sufficient for characterization of a sulfonylurea drug, since the kinetic binding parameters may also determine its acute blood sugar lowering efficacy.

Calcitonin receptor in human placental syncytiotrophoblast brush border and basal plasma membranes

The physiology of calcium transport through the placenta has not been studied thoroughly. In particular, the effect of calcaemic hormones on this process has never been reported. In this paper we questioned if calcitonin, a hypocalcaemic hormone, is also implicated in the regulation of calcium transport by one of the placental syncytiotrophoblast bipolar membranes. In order to investigate the implication of calcitonin on calcium transport, we first studied whether this hormone binds to any of these bipolar membranes i.e. purified syncytiotrophoblast brush border (facing the mother) and basal plasma membranes (facing the fetus). The initiation of binding of human [ 125I]calcitonin to the two types of membranes was rapid and reached a steady state after 10 min of incubation at 37°C. The number of binding sites and the affinity of these receptors for the hormone were studied for each type of membrane, with concentrations of [ 125I]calcitonin varying from 0.01 to 1.8 nM. Scatchard analysis revealed a single affinity binding site for human calcitonin with K d s of 0.83 ± 0.09 nM and 0.67 ± 0.26 nM for brush border and basal plasma membranes respectively. The maximal number of receptors was significantly different ( p < 0.001) in the two membranes: B max of 66.64 ± 9.15 fmol/mg protein for brush border membranes and 19.66 ± 2.80 fmol/mg protein for basal plasma membranes. Competitive displacement of [ 125I]calcitonin with other ligands showed the following potencies between human calcitonin > salmon calcitonin > calcitonin gene-related peptides and segments analogues but no competition with some human calcitonin gene-related peptides fragments. Half-maximal displacement concentration for human calcitonin was reached at approximatively 1 nM for BBM and 0.1 nM for BPM. Calcitonin stimulated inositol phosphate production in both membranes by 175% and 330% in BBM and BPM, respectively. We conclude that calcitonin receptors are present in the two polar syncytiotrophoblast membranes, but are more abundant in brush border membranes, facing the mother. These results also suggest that in the placenta, both maternal and fetal calcitonin might intervene in ion transport and particularly calcium transport or on the intracellular metabolism of the placental syncytiotrophoblast cells.

Luminal Ca2+ increases the affinity of inositol 1,4,5-trisphosphate for its receptor

Luminal Ca2+ has been proposed to regulate the sensitivity of intracellular Ca2+ stores to InsP3 and perhaps thereby to contribute to the mechanisms responsible for regenerative intracellular Ca2+ signals. Since Ca2+ release from intracellular stores is accompanied by K+ influx into the stores, InsP3-stimulated Ca2+ mobilization can be inhibited by substantially reducing the [K+] of incubation media. By measuring [3H]InsP3 binding to permeabilized hepatocytes in K(+)-deficient media, thereby preventing InsP3-stimulated Ca2+ mobilization, we have examined the effects of Ca2+ within the intracellular stores on equilibrium binding of InsP3 to its receptor. Our results demonstrate a small, but significant (P < 0.05, n = 8-9), effect of luminal Ca2+ on InsP3 binding; the concentration of InsP3 causing half-maximal displacement of [3H]InsP3 (1.25 nM) was 3.5 nM for empty stores and 2.1 nM for stores containing Ca2+. Our results suggest that at least part of the stimulatory effect of luminal Ca2+ on InsP3-stimulated Ca2+ mobilization may result from an effect of luminal Ca2+ on InsP3 binding to its receptor.

Features of insulin receptor interaction in placenta from normal, overt and poorly controlled gestational diabetic patients.

Features of insulin binding to trophoblast plasma membranes were studied in six normal pregnant women (NP), six overt diabetes (ODP) and six poorly controlled glycemic gestational patients (PCDP) i.e. women who did not strictly follow the management of diabetes mellitus during pregnancy. A decreased maximum specific insulin receptor binding per 0.1 mg membrane protein in placenta from PCDP (12%) was found comparing with that from ODP or NP (17.5% and 36.2%, respectively, P < 0.01), The insulin binding in PCDP declined at a faster rate until it reached minimum when studied at a higher temperature (25-37 degrees C). The binding equilibrium was likewise attained faster at this temperature than that at lower temperature of 4 degrees C for all studied groups. The insulin receptor binding in all studied groups was pH dependent. The maximum binding in ODP and PCDP groups was attained at pH 7.8 while for NP maximum binding was at pH 7.4. The competitive binding assay was carried out with 14 concentrations of unlabelled insulin and the half maximal displacement of 125I-insulin was at 8 x 10(-9) M, 6 x 10(-9) M and 4 x 10(-9) M for NP, ODP and PCDP, respectively (P < 0.05) suggesting the differences in the effect of glycemic control on the insulin binding. Furthermore the binding yielded curvilinear Scatchard plots with the apparent affinity of the receptors being affected in the ODP and PCDP groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor I: a mitogen for rat Schwann cells in the presence of elevated levels of cyclic AMP.

To develop effective procedures for improving the regeneration of peripheral nerves and for preventing the formation of neurofibromas, it is necessary to identify the different mitogens that stimulate the proliferation of Schwann cells. Insulin-like growth factor I (IGF-I), which is a potent autocrine growth factor in many tissues, is synthesized by proliferating Schwann cells. However, the role of IGF-I in stimulating their division is still uncertain. Here we show that nanomolar concentrations of IGF-I stimulate the growth of Schwann cells in primary culture. IGF-I alone was uneffective, but in the presence of forskolin (5 microM) or dibutyryl cyclic AMP (dbcAMP, 10 microM), it became a potent mitogen. Neither IGF-II nor epidermal growth factor (EGF) were effective even in the presence of forskolin. Insulin also stimulated Schwann cell proliferation in the presence of forskolin, but only at micromolar concentration. Receptors for IGF-I were visualized on the Schwann cell surface by indirect immunofluorescence staining using anti-human IGF-I receptor antibodies. Their presence was also assessed by binding assays using [125I]-IGF-I as a ligand. Scatchard analysis showed a single class of high-affinity receptors (Kd = 1.5 nM). Competitions studies with unlabeled IGF-I or insulin indicated a half-maximal displacement of [125I]-IGF-I by IGF-I at about 5 nM, while insulin was about 500-fold less effective. The number of binding sites for IGF-I was increased by exposing cells for 3 days to forskolin (-forskolin: about 5,100; + forskolin: about 12,200 binding sites/cell).(ABSTRACT TRUNCATED AT 250 WORDS)