Radiolabelled Antagonist Research Articles

Conus venom interaction with α 2-adrenergic receptors in calf retina membranes

α 2-Adrenergic receptors were identified in calf retina membranes by the specific binding of the radiolabelled antagonist [ 3H]RX 781094. Crude venoms from various Conus species did not interact with the radioligand but were able to inhibit radioligand binding to the α 2-receptors with the following order of potency: C. planorbis (IC 50 = 2.1 μg protein/ml) ≈ C. tessulatus (IC 50 = 2.7) > C. eburneus (IC 50 = 19) > C. textile (IC 50 = 54) > C. geographus (IC 50 = 130). Venom from 17 other species was less or not active at all. Venom competition binding curves were steep and not affected by GTP. In contrast, the ( − )-epinephrine competition binding curve was shallow and underwent a rightward shift and steepening in the presence of GTP. The venom-α 2-receptor interaction was completely inhibited by the calcium chelating reagent EGTA. These data suggest that the venom of certain Conus species contains peptide toxins which are capable of shielding the binding site of α 2-receptors in an antagonistic manner.

Two-step isomerization of quinuclidinyl benzilate-muscarinic receptor complex

A kinetic analysis was made of the dissociation reaction of the muscarinic receptor- l-[ 3H]quinuclidinyl benzilate complex at 25°C in 0.05 M K-phosphate buffer. The course of the reaction was followed by the decrease in the concentration of the membrane-bound radiolabelled antagonist while rebinding was prevented by the excess of nonradioactive quinuclidinyl benzilate. It was found that both bi- and mono-exponential kinetic curves of the process can be observed, depending on the time moment when the dissociation reaction is started. If the receptor-ligand complex had been incubated for a sufficiently long time before the excess of the nonradioactive ligand was added to “displace” the radioactive ligand from the complex, the dissociation reaction followed the first-order kinetics. The bi-exponential kinetics of the dissociation process was obtained if the displacement was started within a short time interval after the complex formation between the receptor and l-[ 3H]quinuclidinyl benzilate. The data obtained were analysed within the framework of a reaction scheme containing two consecutive isomerization steps of the receptor-antagonist complex. The “isomerized” receptor-ligand complexes differ in their dissociation rate and therefore their interconversion changes the observed kinetic behaviour of the dissociation reaction of the receptor-ligand complex.

Agonist/N-ethylmaleimide mediated inactivation of β-adrenergic receptors: Molecular aspects

Incubation of turkey erythrocyte and rat lung membranes with the β-adrenergic agonist isoproterenol in the presence of the group specific reagent N-ethylmaleimide (NEM) causes the apparent inactivation of a well defined fraction of the β-adrenergic receptors. This inactivation process requires the coupling between the receptors and the guanine nucleotide binding regulatory component of the adenylate cyclase system. Accordingly, only the isoproterenol/NEM sensitive receptor population is susceptible to undergo this functional coupling. Incubation of the treated membranes in alkaline buffer and/or in the presence of GTP reactivates the receptors. The reactivated receptors show the initial binding properties for both isoproterenol and the radiolabelled antagonist [ 3H]-dihydroalprenolol and are still dithiothreitol sensitive. Moreover the reactivated receptors can again couple to the regulator in the presence of agonists. The present study makes it possible to discriminate between the different models advanced for the N-ethylmaleimide action and is valid for β 1- as well as β 2-adrenergic receptors. The reagent appears indeed to freeze the hormone-receptor-regulator complex by alkylating the latter component, as previously proposed by Korner et al. (1982) J. biol. Chem. 257, 3389–3396. It is also suggested that the existence of a limited fraction of coupling-prone receptors results from differences in the membrane microenvironment or from a structural heterogeneity of the receptors rather than from a stoichiometric limitation of the regulator concentration.

Agonist mediated conformational changes of solubilized calf forebrain muscarinic acetylcholine receptors

Muscarinic receptors in calf forebrain membranes can be identified by the specific binding of the radiolabelled antagonist [ 3H]dexetimide. These receptors (2.8 pM/mg protein) comprise two non-interconvertible subpopulations respectively high and low agonist affinity but with the same antagonist affinity. For all the agonists tested the low affinity sites represent 85±5% of the total receptor population. 0.5% Digitonin solubilized extracts contain 0.8 pM muscarinic receptor/mg protein. In contrast with the membranes, these extracts contain only sites with low agonist affinity. The alkylating reagent N-ethylmaleimide causes an increase of the acetylcholine affinity for the low affinity sites in membranes as well as for the solubilized sites. This effect is time dependent until a maximal 3-fold increase in affinity is attained. The rate of N-ethylmaleimide action is enhanced by the concomitant presence of agonists. In contrast, N-ethylmaleimide does not affect antagonist binding. This suggests that agonists mediate a conformational change of both the membrane bound low affinity muscarinic sites and of the solubilized sites, resulting in their increased susceptibility towards NEM alkylation.

Muscarinic Acetylcholine Receptors in Torpedo Electric Organ: Effect of Guanine Nucleotides

The effect of guanine nucleotides on the binding properties of presynaptic muscarinic receptors has been studied in a membrane preparation from the electric organ of Torpedo marmorata by measuring the competitive displacement of the radiolabelled antagonist, [3H]quinuclidinyl benzilate, by nonradioactive muscarinic ligands. The binding of the antagonists, atropine, scopolamine and pirenzepine was to a single class of sites [slope factors (pseudo Hill coefficients) close to 1] and was unaffected by 0.1 mM GTP. The binding of the N-methylated antagonists, N-methylatropine and N-methyl-scopolamine was more complex (slope factors less than 1) but also insensitive (N-methylatropine) to 0.1 mM GTP. Agonist binding was complex and could be resolved into two binding sites with relatively high and low affinities. The proportion of high-affinity sites varied with the nature of the agonist (15-80%). Agonist binding was depressed by 0.1 mM GTP, and the order of sensitivity was oxotremorine-M greater than carbamoylcholine greater than muscarine greater than acetylcholine greater than arecoline greater than oxotremorine. The binding of pilocarpine, a partial agonist, was unaffected by GTP. With carbamoylcholine as a test ligand the GTP effect on agonist binding was half-maximal at 12 microM. GDP and guanylylimidodiphosphate produced comparable inhibition of carbamoylcholine binding, but GMP and cyclic GMP were ineffective, as were various adenine nucleotides. Analysis of agonist binding in terms of a two-site model indicates that the predominant effect of guanine nucleotides is to reduce the number of sites of higher affinity.

The β-adrenergic receptor in mammalian placenta: Species differences and ontogeny

Summary β-Adrenergic receptors were demonstrated in placental membrane fractions of five mammalian species (rat, rabbit, guinea pig, sheep and human) with the radiolabelled antagonist (−)-[3H]-dihydroalprenolol ((−)-[3H]DHA). Identical membrane preparations showed a variable maximum binding capacity in different species, ranging from 70±6.7 in the term rat to 315 ±28.2 in the term rabbit. Binding affinity did not change with species and (−)-[3H]DHA binding was associated with a catecholamine-sensitive adenylate cyclase in each. In the rabbit placenta, β-adrenergic receptor subtypes were partially characterized and the ontogeny of the placental β-receptor demonstrated. Agonist competition for the (−)-[3H]DHA binding site showed the order of potency (−)-isoproterenol≫ (−)-epinephrine≫ (−)-norepinephrine characteristics of a β2 subtype. Guanine nucleotides (Gpp(NH)p) significantly reduced agonist affinity for the binding site and increased the Hill coefficient from 0.6 to approximately 1.0, as expected in a ‘coupled’ β-adrenergic receptor. Competition between (−)-[3]DHA and β-adrenergic receptor subtype selective agents was performed to define the β-receptor subtype populations. In contrast to the linear Hofstee plots obtained with classical agonists, curvilinear Hofstee plots were obtained with both metoprolol (β1 selective) and zinterol (β2 selective), indicating the presence of two distinct receptor subtype populations. A predominance of the β2 subtype was demonstrated with both agents in the term rabbit placenta (35 per cent β1, 65 per cent β2). β-Adrenergic receptor density in rabbit placentae from 16 to 30 days' gestation was determined. Protein and DNA content were also determined in these placentae. A period of growth by cellular hyperplasia from day 16 to day 25, during which DNA increased threefold and protein per DNA remained constant, was followed by a period of growth by cellular hypertrophy from day 25 to day 30, during which DNA remained constant and protein per DNA increased twofold. The β-adrenergic receptor density increased markedly with gestation (181±29 to 330±37 fmol/mg). There was no change in binding affinity with gestation. β-Receptor density per DNA remained constant during the period of cellular hyperplasia, increased twofold during early cellular hypertrophy (day 25 to day 27) and then remained constant to term (day 30). An adenylate cyclase coupled β-adrenergic receptor is present in the placentae of several mammalian species and changes in density in parallel with maturational events in one species, suggesting a role for catecholamines in placental function.

Competitive binding studies with multiple sites: Effects arising from depletion of the free radioligand

Apparently anomalous behaviour arises if a radiolabelled drug and a non-radioactive drug compete for binding to a membrane-bound receptor when (a) there is severe depletion of the radiolabelled drug and (b) the non-radioactive drug binds to a heterogeneous population of binding sites. In extreme cases, binding of the non-radioactive drug to the sites of high affinity can be obscured completely. This phenomenon is illustrated by the binding of carbachol to muscarinic receptors, estimated by inhibition of a radiolabelled antagonist.