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At least 793 records · Page 44Linked to original sources
PubMed1985

Evaluation of a photolabile derivative of 1,25-dihydroxyvitamin D3 as a photoaffinity probe for 1,25-dihydroxyvitamin-D3 receptor in chick intestinal cytosol.

We evaluated the viability of 1 alpha, 25-dihydroxyvitamin D3-3 beta-[N-(4-azido-2-nitrophenyl)glycinate] (1,25-(OH)2-D3-ANG), an analog of 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2-D3) as a photoaffinity probe for 1,25-(OH)2-D3 receptor in chick intestinal cytosol. A competitive-binding assay revealed that chick intestinal cytosolic 1,25-(OH)2- D3 receptor bound to 1,25-(OH)2-D3-ANG approximately 20-times less effectively than it did to 1,25-(OH)2-D3. Irradiation of 1,25-(OH)2-D3- ANG in the presence of chick intestinal cytosolic preparation significantly diminished subsequent binding to 3H-1,25-(OH)2-D3, suggesting that the photoaffinity analog was covalently attached to the receptor. Therefore the nitroarylazide derivative of 1,25-(OH)2-D3 may be a valuable photoaffinity probe for the characterization of the 1,25-(OH)2-D3 receptor.

Affinity Labels↗
PubMed1986

Madin-Darby canine kidney cells display type I and type II insulin-like growth factor (IGF) receptors.

Using affinity cross-linking techniques, we report the presence of type I IGF and type II IGF receptors in Madin-Darby canine kidney cells, a line of cells lacking insulin receptors. The IGF receptors were further characterized by competition binding studies and found to be similar to IGF receptors in other tissue types. In Madin-Darby canine kidney cells, the type I IGF receptor binds IGF-I greater than IGF-II greater than insulin and the type II IGF receptor binds IGF-II and IGF-I with approximately the same affinity, but does not bind insulin.

Animals↗
PubMed1991

Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells.

VIP receptors on AR42J rat pancreatic cells were analyzed by competition binding, affinity labeling and by N-glycanase digestion analyses. These studies revealed the presence of specific, high affinity (Kd approximately 1 nM) VIP receptors with a mass of 67 kDa or 59 kDa under reducing or non-reducing conditions, respectively. N-glycanase digestion of affinity labeled membranes generated a core receptor protein of approximately 44 kDa and evidence for at least two N-linked glycans on the mature receptor. The receptor lacked O-linked oligosaccharides but contained terminal sialic acid residues on its N-linked glycan(s) based on digestions with O-glycanase and neuraminidase. The similarity of the AR42J VIP receptor to the recently cloned cDNA for human VIP receptors makes this cell line an attractive model for further analysis of VIP receptor signal transduction events.

Animals↗
PubMed1991

Antigen specificity of anticolipase monoclonal antibodies: characterization of colipase-Fab complexes using gel filtration high-performance liquid chromatography.

The epitope specificity of eight mouse monoclonal antiporcine procolipase antibodies (MAbs) was characterized on the basis of their competitive binding with antigen. Binary and ternary Fab-colipase complexes formed between antibody and porcine procolipase or its trypsin activated derivative were identified using gel filtration HPLC. The eight MAbs were divided in two groups that recognized overlapping epitopes located in distinct antigenic regions on procolipase. The gel filtration HPLC technique allowed to characterize two MAbs which did not react with solid-phase coupled antigen. Three MAbs formed Fab-antigen complexes with procolipase and not with activated colipase which suggests that epitopes recognized by these MAbs involve residues of the N-terminal pentapeptide of procolipase.

Animals↗
PubMed1992

Concomitant expression of receptor subtype and isopeptide of endothelin by human adrenal gland.

We studied whether specific receptors for endothelin (ET) isopeptide exist in human aldosterone-producing adenoma and normal adrenal cortex, and whether ET isopeptides are produced by human adrenal gland. Competitive binding studies using [125I]ET-1 as a radioligand revealed the presence of a single class of high-affinity binding sites for ET-1 with the apparent KD of 70 +/- 31 pM and Bmax of 226 +/- 139 fmol/mg protein in adenoma membranes almost comparable to those in adjacent normal cortex. The apparent Ki for ET-2 and ET-3 were 89 +/- 33 pM and 82 +/- 16 pM, respectively. Northern blot analysis of poly(A)+ RNA of adenoma and adjacent normal cortex using cDNAs for ET receptor subtype (ETA, ETB) and ET isopeptide (ET-1, ET-3) as probes revealed that ETA and ETB receptors as well as ET isopeptides (preproET-1, preproET-3) are concomitantly expressed in both tissues. Our data demonstrate for the first time that ET receptor subtype (ETA and ETB) and ET isopeptide (ET-1 and ET-3) are concomitantly expressed by human adrenal cortex, suggesting the potential role of ETs as a local mediator in human adrenal gland.

Adenoma↗
PubMed1983

Cardiac beta-adrenoceptor modulation by amiodarone.

beta-Antiadrenergic properties are part of the pharmacological characteristics of amiodarone. In the present study, the action of amiodarone on rat-heart beta-adrenoceptors was investigated. [125I]Cyanopindolol (CYP) was used to label beta-adrenoceptors in crude rat-heart microsomes. In competition binding experiments, amiodarone up to 10(-6) M did not displace [125I]CYP from cardiac beta-adrenergic receptors. The effects of amiodarone on the number and affinity for [125I]CYP of beta-adrenoceptors were evaluated in saturation experiments. In vitro exposure of cardiac microsomes to 10(-5) M amiodarone did not modify these parameters. At higher concentrations the beta-receptor number decreased while the affinity for [125I]CYP was not affected. In vivo experiments showed a significant decrease in beta-adrenoceptor density after a single oral dose of 50 mg/kg amiodarone. In chronically treated animals, the same decrease in beta-receptor number was observed 24 hr after the last administration of the drug. 5'-Nucleotidase activity, another specific marker of the plasma membrane, was unaffected by the treatment. These results suggest that part of the beta-adrenergic antagonism of amiodarone is due to a decrease in the beta-adrenoceptor density at the surface of the myocardial cell.

Amiodarone↗
PubMed1987

Two beta-adrenergic pharmacophores on the same molecule. A set of agonist-antagonist combinations.

A series of compounds containing combinations of one or two pharmacophores of the agonist type (isoproterenol) or the antagonist type (propranolol or alprenolol) on the same molecule were prepared. The pharmacophores were connected by a derivative of polyethylene glycol with an average length of six atoms (carbon and oxygen). Furthermore, compounds containing two alprenolol residues, separated by chains of average lengths of 70 or 145 atoms, were synthesized. The abilities of these compounds to interact with beta-adrenoceptors of rat heart and lung tissues were examined by measuring the following parameters: competitive binding with [3H]dihydroalprenolol, activation of adenylate cyclase, and inhibition of isoproterenol-stimulated adenylate cyclase. The affinity of the compound with two isoproterenol pharmacophores for receptor was about the same as that with one isoproterenol pharmacophore and between 30 and 200 times weaker than that of (+/-)isoproterenol. Both mono- and bis-pharmacophore compounds partially stimulated catecholamine sensitive adenylate cyclase and at high concentrations inhibited the stimulation produced by (-)isoproterenol. The affinity of the compound with antagonist (propranolol) and agonist (isoproterenol) pharmacophores on the same molecule was intermediate between that of propranolol and isoproterenol. The compound was only able to inhibit adenylate cyclase activity. Compounds containing two antagonist (alprenolol) pharmacophores bound to receptors with affinities from an order of magnitude lower to about equal to that of the compound containing one pharmacophore. When membranes were preincubated with compounds containing two antagonist pharmacophores and then washed extensively, there were persistent effects of all of these compounds on the binding constants of [3H]dihydroalprenolol. All of these compounds were only able to inhibit adenylate cyclase activity and none exhibited any subtype selectivity at beta-adrenoceptors. The results suggest that, in the beta-adrenergic system, compounds with agonist and antagonist substituents on the same molecule exhibit properties of the substituent with the higher affinity for beta-adrenoceptor, and no agonist activity is evident when two antagonist pharmacophores are linked on the same molecule. All of the above results may be explained without recourse to cross-linking of beta-adrenoceptors with two pharmacophores, a phenomenon cited in similar studies of receptors for opiates and gonadotropin-releasing hormone.

Adrenergic beta-Agonists↗
PubMed1989

Use of N-ethylmaleimide to directly determine the proportion of beta-adrenergic receptors exhibiting high affinity for agonists on human mononuclear leukocyte membranes.

Following incubation with the beta-adrenergic agonist isoproterenol (ISO) and the alkylating agent N-ethylmaleimide (NEM), the number of beta-adrenergic receptors measured on human mononuclear leukocyte (MNL) membranes decreased. Pretreatment induced an approximately 40% loss of measurable receptors. The proportion of receptors lost was that which formed the ternary complex of agonist-receptor-Gs protein (H-R-Gs) and exhibited high affinity for agonists. In comparison studies, the loss of receptors was found to be similar to the proportion of receptors that form the high affinity state of the receptor (%RH) for agonist in agonist competition binding studies. Acute exposure to agonist induced a similar reduction in ISO/NEM "sensitive" receptors and the %RH. These experiments indicate that pretreatment of MNL membranes with ISO/NEM is a powerful tool with which to directly determine the percentage of receptors able to form an agonist high affinity state.

Binding, Competitive↗
PubMed1992

Homoanatoxin: a potent analogue of anatoxin-A.

The natural toxin anatoxin-a (AnTx) is a potent nicotinic agonist that is valuable for the study of nicotinic receptors. We have synthesized 2-(propan-1-oxo-1-yl)-9-azabicyclo[4.2.1]non-2-ene, the homologue of AnTx in which the side-chain is extended by one methylene unit from a methyl to an ethyl ketone. This chemistry would allow the generation of a tritiated product and the homologue, designated homoanatoxin (HomoAnTx), has been characterized here with that aim in mind. In competition binding assays at neuronal nicotinic ligand binding sites characterized by [3H]nicotine and [125I]-alpha bungarotoxin, HomoAnTx retained the same potency as the parent molecule, with Ki values of 7.5 nM and 1.1 microM, respectively. In contrast, it showed little inhibition of muscarinic binding defined by [3H]-quinuclidinyl benzilate. HomoAnTx is a potent nicotinic agonist in frog muscle contracture assays, having four times the potency of carbamylcholine and one tenth of the activity of AnTx itself. The N-methylated version of HomoAnTx was more than two orders of magnitude weaker in both functional and binding assays. The successful synthesis of HomoAnTx with retention of high nicotinic potency offers a route for the generation of novel, potent radiolabelled nicotinic ligands.

Animals↗
PubMed1979

Nicotinic cholinergic receptors in brain synaptosomes.

In order to elucidate pharmacological characteristics of nicotinic receptors in the brain, competitive binding of nicotine analogues or cholinergic agents to brain particles was studied utilizing [3H]nicotine. The binding of [3H]nicotine to brain crude nitochondrial or synaptosomal fraction was progressively inhibited by the addition of increasing amounts of native nicotine or nornicotine, but cotinine had little effect. Of the myelin, synaptosomal and mitochondrial subfractions of the crude mitochondrial fraction, [3H]nicotine binding was almost exclusively confined to synaptosomes. In this binding, the affinity constant was 1.97 X 10(9)/M and the maximum binding capacity was 7.30 X 40(-8) M/g of protein. This binding was reduced by 50% in the presence of 4 X 10(-7) M D-tubocurarine and 10(-6) M carbamylcholine, while atropine was the least effective of the drugs tested. These findings suggest the possible existence of nicotinic cholinergic receptors in the synaptic region. A study of regional differences in nicotinic cholinergic receptors showed that higher specific bindings of nicotine to synaptosomes occurred in the hypothalamus, hippocampus and thalamus. These findings suggest that the nicotinic cholinergic mechanism plays an important role in these regions of the central nervous system.

Animals↗
PubMed1991

Characterization of muscarinic receptor subtype of rat eccrine sweat gland by autoradiography.

The muscarinic cholinergic receptor of rat eccrine sweat gland was characterized using quantitative autoradiography and [3H]QNB as radioligand. The distribution of radioligand was maximal in the secretory coil. Autoradiographic competition binding studies were performed using selective antagonists to M1 (pirenzepine), M2 (AF-DX 116), and M3 (4-DAMP) and the classical nonselective antagonist atropine. pKi for pirenzepine, AF-DX 116, 4-DAMP, and atropine was 6.58, 5.47, 8.50, and 8.66 respectively indicating that the eccrine sweat gland muscarinic receptor was predominantly M3.

Animals↗
PubMed1988

Effect of monovalent cations on polyvalent cation-induced fusion of phosphatidylserine small unilamellar vesicles.

Fluorescence internal contents mixing assay was used to monitor the fusion of phosphatidylserine (PS) small unilamellar vesicles, initiated by metal ions (Ca2+, La3+ and Tb3+), at various concentrations of monovalent cations (Li+, Na+ and K+). The influence of ionic strength (0.02-1.0 M) on the threshold concentration of "fusogenic" cations required to induce fusion was measured. The threshold concentrations increased monotonically (1 mM at 0.1 M to 3.1 mM at 1 M) with the increasing ionic strength of the solution for Ca2+, but remained unchanged for both La3+ and Tb3+. Changes in the ionic strength of the encapsulated solution did not alter the threshold concentrations for all the ions studied, in the range 0.02-0.3 M. The results are analyzed in terms of competitive binding between the monovalent ions and the "fusogenic" ions (Ca2+, Tb3+ and La3+). It is shown that there is a critical value for calcium bound-PS, below which no massive fusion occurs. Bound and free fractions of PS are calculated based on the Gouy-Chapman model, taking activities rather than concentrations of metal ions into account. Our experiments also show that monovalent ions alone do not induce fusion even at high concentrations.

Animals↗
PubMed1979

The combined radioimmunoassay of triiodothyronine and thyroxine.

An RIA method for the concurrent determination of serum triiodothyronine (T3) and thyroxine (T4) is described. The extraction of the hormones from serum and competitive binding reactions using specific T3 and T4 antisera were conducted in sequence in a single Sephadex column. Precision studies resulted in average interassay relative standard deviations for T3 and T4 of 17 and 9.5%, respectively. The assay also exhibited satisfactory recovery and correlation with reference procedures (T3: y = 0.84x -- 36, r = 0.98; T4: y = 0.96x + 0.06, r = 0.98, where y is the value obtained by the combined method and x is the value obtained by the reference method.

Binding, Competitive↗
PubMed1984

Quantitative determination of immunoglobulins in the cerebrospinal fluid based on their light chain properties using a solid-phase enzyme immunoassay.

A method for quantitative determination of immunoglobulins in the cerebrospinal fluid (CSF) on the basis of their light chain properties is described. The method is a solid-phase enzyme immunoassay, based upon competitive binding of immunoglobulins in unknown and labeled immunoglobulin for antibody adsorbed to the wells of a microplate. The values for immunoglobulins kappa (Ig-k), lambda (Ig-l) and k/l ratio obtained from 38 subjects without neurological disorders were 8.6 +/- 4.0 micrograms/ml, 7.0 +/- 2.6 micrograms/ml and 1.21 +/- 0.21, respectively (mean +/- SD). Our method is considered to be less influenced by the molecular size and weight of immunoglobulins than radial immunodiffusion, and thus may provide more precise values of Ig-k and Ig-l.

Adolescent↗
PubMed1991

A new immunochemical assay for biotin.

A double antibody technique has been developed to separate free biotin from bound biotin after competitive binding of [3H]biotin and unlabelled biotin to avidin. Antiavidin goat antibody was added followed by the addition of antigoat IgG antibody linked to agarose. Centrifugation separated the free biotin from the biotin bound to the avidin complex. The method was suitable for the detection of the amounts of biotin contained in 100-200 microliters of plasma or 5-10 microliters of urine. Normal values for the concentration of biotin in plasma and urine determined by this assay were 1.27 +/- 0.67 nmol/l and 49.1 +/- 35.7 mumol/mol creatinine, respectively.

Animals↗
PubMed1990

Characterization of alpha 2-adrenoceptors in the rat: proximal tubule, renal membrane and whole kidney studies.

In the present study, alpha 2-adrenoceptors have been characterized in rat renal proximal tubules which were isolated by a Percoll gradient technique. Competitive binding curves with [3H]rauwolscine (0.5 nM) and alpha 2-adrenoceptor agonists and antagonists were consistent with an alpha 2B-adrenoceptor subtype. However, the rank order of potency (Ki) for clonidine and UK 14,304 was reversed from that reported for other tissues (clonidine, 48 nM greater than UK 14,304, 330 nM). This rank order was confirmed in a crude renal membrane preparation consisting of whole kidney as well as separated medullary and cortical segments. An intrarenal infusion of clonidine at 11 nmol/kg per min resulted in a greater diuresis and natriuresis than an equimolar dose of UK 14,304 suggesting that clonidine also had a greater affinity in the collecting tubules. Further displacement studies in proximal tubules with [3H]rauwolscine and calcium channel blockers demonstrated that verapamil was the most potent (Ki, 2.3 microM), followed by diltiazem (48% displacement at 100 microM) and then nifedipine (no displacement at 100 microM). These studies indicated that alpha 2-adrenoceptor in the rat proximal tubule may be of the alpha 2B-adrenoceptor subtype. Further studies will be required to determine whether the reverse rank order of potency of clonidine and UK 14,304 is consistent with an alpha 2-adrenoceptor subtype which is different from that found in other tissue.

Animals↗
PubMed1992

Evidence for more than two central benzodiazepine receptors in rat spinal cord.

Competitive binding assays were performed in rat spinal cord membranes at 0 degrees C against [3H]Ro15-1788. The displacement curves of Ro15-1788 and alpidem produced pseudo-Hill slopes of 1.0 and 0.5, respectively. In addition, 5-10% of the specifically bound [3H]Ro15-1788 could not be displaced by greater than 100 microM of alpidem. These two pieces of evidence strongly suggest the presence of at least three central benzodiazepine binding sites in the spinal cord.

Animals↗
PubMed1993

High- and low-affinity states of dopamine D1 receptors in schizophrenia.

Using SKF-38393/[3H]SCH-23390 dopamine D1 receptor agonist/antagonist competition binding technique, we showed that schizophrenics distinguished from the neuro-psychiatric and normal controls by exhibiting a significant increase in the density of the low-affinity state, and a concomitant enhancement in the binding affinity of the high-affinity state of D1 dopamine receptors in caudate nucleus. The results suggest that perturbation in dynamic equilibrium of high- and low-affinity states of D1 receptors underlies the pathogenesis of schizophrenia.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗