Effects of androgen on alpha- and beta-adrenergic receptors in membranes from the rat seminal vesicle.

The effects of castration and androgen-replacement on adrenergic receptors in membranes from the rat seminal vesicle were studied. Membranes from seminal vesicles showed saturable and high-affinity binding sites for the beta-adrenergic receptor antagonist, [3H]dihydroalprenolol ([3H]DHA), and the alpha 1-adrenergic receptor antagonist, [3H]prazosin. Castration markedly reduced beta-adrenergic receptors with decreasing the effect of GTP modulating the receptor-ligand affinity, suggesting defects in both the receptor per se and the guanine-nucleotides-regulating mechanism after castration. In contrast, castration increased alpha 1-adrenergic receptors and androgen-replacement reversed this change. The effects of GTP decreasing the alpha 1-receptor binding affinity to the radioligand were observed to a similar extent in the castrated and control membranes. These results demonstrate an inverse regulation by androgen on beta- and alpha 1-adrenergic receptors in membranes of the rat seminal vesicle.     

Utility of Polycation-Treated Filters for the Assay of Receptors for VIP

Abstract

This study examined the utility of four polycationic agents for treating glass fibre filters used in the receptor binding assay for vasoactive intestinal peptide (VIP). Polyethylenimine (PEI), polybrene, protamine and methylated bovine serum albumin proved satisfactory in terms of low filter binding of free radioligand and retention of membrane-bound radioligand. Their performance was superior or comparable to untreated Millipore EGWP cellulose acetate filters which we had previously utilized but which are no longer manufactured. The results with polycations indicate the importance of ionic interactions between filter, biological membranes and radioligand in determining the performance of a filtration assay for radioligand-receptor binding. At a practical level, PEI has the disadvantage of potential toxicity. The satisfactory performance of the other polycations indicates that they provide safer alternatives to PEI for filtration assay of the VIP receptor and possibly receptors for other basic ligands.     

Identification of an atrial natriuretic peptide specific receptor in human kidney

A specific receptor for human atrial natriuretic peptide (h-ANP) was identified in the human kidney using the radioligand binding assay. Samples were prepared from non-malignant renal tissues obtained at nephrectomy of patients with renal carcinoma. Binding studies using [125I]hANP were performed at 0 degree C for 20 minutes and terminated by a rapid filtration technique. Scatchard plot analysis revealed [125I]hANP bound to a single class of binding site (Kd = 0.4 +/- 0.2 nM) with a density of 16 +/- 4 fmol/mg protein in the renal cortex (n = 7). The binding was rapid and maximal binding was obtained within 20 minutes after the start of incubation. Radioligand displacement was observed in a dose dependent fashion when cold hANP was entered into the reaction mixture. However, unrelated agents, such as angiotensin II or 1-epinephrine, did not affect the binding. This is the first time characterization of the hANP receptor in the human kidney has been conducted using a Scatchard plot analysis.     

Beta-adrenergic receptors in early human placenta characterization of [3H]-dihydroalprenolol binding

The binding characteristics of beta-adrenergic ligand [³H]-dihydroalprenolol (DHA) were determined in particulate membranes of early human placenta (8 – 12 weeks of gestation). [³H]-DHA binding to crude membrane fractions was rapid, reversible, saturable and linearly correlated with the membrane protein concentration. Scatchard analysis of saturation experiments showed a K_D of 2.80 ± 0.9 nM and a density of binding sites of 330.30 ± 93.5 fmol/mg protein. Agonist potency isoproterenol epinephrine norepinephrine indicated that early human placenta contains an adrenergic receptor of beta-2 subtype.     

The role of cytosolic and membrane factors in processing of the human beta-2 adrenergic receptor following translocation and glycosylation in a cell-free system.

The beta-2 adrenergic receptor has been proposed to have seven membrane-spanning domains. Expression of functional beta-2 adrenergic receptor was achieved in a heterologous cell-free system composed of rabbit reticulocyte lysate and microsomal membranes from Xenopus laevis oocytes. The functional state of the receptor protein can be determined by ligand-binding assays and by the ability of ligands to alter the susceptibility of the receptor to proteinase K digestion. The process by which functional receptor is made was studied. The receptor protein remains nonfunctional immediately following translocation and glycosylation, and additional processing steps are needed before the receptor is able to interact with ligands. These processing steps require intact microsomal membranes as well as several cytosolic factors including ATP and one or more high molecular mass (greater than 30 kDa) factors but do not require receptor glycosylation and are not inhibited by nonhydrolyzable GTP analogues.     

Biophysical characterization of the purified alpha 1-adrenergic receptor and identification of the hormone binding subunit

The alpha 1-adrenergic receptor has been solubilized in active form from rat hepatic membranes with the nonionic detergent, digitonin, and purified by affinity and gel filtration chromatography to homogeneity with a specific activity of 14,400 pmol/mg of protein. The affinity chromatographic steps of the purification procedure were achieved by the use of a newly synthesized analog (2-[4(2-succinoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, CP-57,609) of the highly selective alpha 1-adrenergic antagonist, prazosin, immobilized via an amide linkage to agarose. The resulting purified receptor bound [3H]prazosin and a variety of adrenergic agents with the specificity, stereoselectivity, and affinities equivalent to those observed with membrane-bound and solubilized receptor preparations. The purified receptor.digitonin complex had a Stokes radius of 49 A and a sedimentation coefficient (s20w) of 7.1, as determined by AcA-34 gel filtration chromatography and sucrose gradient density centrifugation, respectively. Based on these hydrodynamic parameters, the calculated molecular weight of the receptor.digitonin complex was estimated at 147,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis following the final purification step revealed a single band of protein at 59,000 daltons from which [3H]prazosin binding activity could be recovered after renaturation of the receptor protein. These findings indicate that the protein purified from rat hepatic membranes is the hormone binding component of the alpha 1-adrenergic receptor and that the receptor molecule most likely contains more than one Mr = 59,000 subunit.     

Ontogeny of alpha 1- and beta-adrenergic receptors in rat lung

The binding characteristics of the alpha 1-selective adrenergic ligand [3H]-prazosin were determined in particulate membranes of rat lung from day 18 of gestation to adulthood. Specific binding was present at all ages studied, was reversible and inhibition of specific binding by agonists followed the order of potency: (-)-epinephrine = (-)-norepinephrine much greater than (-)-isoproterenol greater than (+)-norepinephrine. Inhibition by antagonists followed the order of potency: prazosin greater than WB4101, much greater than yohimbine. Binding capacity increased during the neonatal period from 52 +/- 9 fmoles x mg-1 protein in lung preparations on day 18 of a 21 day gestation increasing to 105 +/- 4 fmoles x mg-1 protein (mean +/- SE) by postnatal day 15. Binding activity decreased thereafter, reaching adult levels by 28 days of postnatal age, 62 +/- 3 fmoles x mg-1 protein. This pattern of alpha 1-adrenergic receptor density was distinct from that of beta-adrenergic receptors identified in rat lung membrane with the beta- adrenergic antagonist, (-)-[3H]dihydroalprenolol ((-)-[3H]DHA). (-)-[3H]DHA binding increased dramatically during this same time period, from 46 +/- 4 fmoles x mg-1 protein on day 18 of gestation to 496 +/- 44 fmoles x mg-1 protein in the adult lung. Affinity for [3H]-prazosin and (-)-[3H]DHA did not change with age. Pulmonary alpha 1-adrenergic receptors are present as early as 18 days of gestation in the rat and alpha 1-adrenergic receptor density is maximal by 15 days of postnatal age. The timing of the changes in alpha 1-adrenergic receptors correlates with the timing of increased sympathetic innervation of the developing rat lung and is distinct from that of beta-adrenergic receptor sites.     

Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture

A rapid and sensitive filtration assay for quantitating the specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to whole cells in culture is described. Cell monolayers are incubated with [3H]TCDD in the presence or absence of excess unlabeled ligand, detached from the culture dish with trypsin, filtered, and washed with cold (-78 degrees C) acetone to separate free and nonspecifically bound TCDD from specifically bound TCDD. TCDD receptor binding parameters were characterized in the murine hepatoma cell line Hepa1c1c7. The lower limit of detection of TCDD specific binding was in a sample equivalent to 10 micrograms of total cell protein. The equilibrium dissociation constant and stereospecificity for binding to the TCDD receptor were the same as those previously reported with other TCDD receptor assays on broken cell preparations. Analysis of binding in the murine hepatoma TCDD receptor variants TAO-c1BPrc1 and BPrc1 indicated that this assay will detect receptor number or affinity variants, but will not detect nuclear transfer deficient variants. The major advantage of the whole cell binding assay is that it provides the means to rapidly and reproducibly quantitate TCDD specific binding in small samples of whole cells in culture. In addition, this method eliminates loss or degradation of the receptor protein during the fractionation of cells required in previously reported methods. This method should prove useful in screening clonal cell populations for TCDD receptor number and affinity variants, and in screening for TCDD receptor binding activity in complementation studies of receptor deficient cells.     

A radioligand-binding assay for adenosine in tissue extracts

A radioligand-binding assay employing adenine analog-binding protein from rabbit crythrocytes and [³H]adenosine of high specific activity measures less than 1 pmol of adenosine in neutral HClO₄ extracts of tissue. Adenine and its nucleotides interfere with adenosine binding, so are removed by preliminary chromatography on PEI and phenyldihydroboryl cellulose columns. Free and bound adenosine are separated by filtration through cellulose acetate membranes or by absorbing free adenosine on charcoal. The recovery of 1.0–4.0 pmol of adenosine added to blood extracts and carried through purification and radioligand assay averaged 100% with an absolute error of ±0.23 pmol.     

Receptor analysis: An arithmetic correction improves precision and accuracy

Data of receptor analysis by ligand binding experiments should be processed using the formula DCORR = (B1 - B2.F1/F2)/VS.DCORR is an estimate of the concentration of receptor-bound radioligand; B1 and F1 are estimates of bound and free radioligand in assay 1; B2 and F2 are the corresponding values obtained from the parallel assay 2, which contains an additional excess of nonlabeled ligand; VS is the volume of assays 1 and 2 that was submitted to separation. DCORR will be superior to the conventional formula, D = (B1 - B2)/VS, if the radiolabeled receptor-ligand complexes are incompletely separated from nonspecifically bound and free radioligands. DCORR corrects for the systematic underestimation of the specifically bound radioligand implicated in D as well as for random errors due to imprecise pipetting during preparation of the parallel assays. The superiority of DCORR over D is verified by processing the data of androgen receptor analyses using agar gel electrophoresis for separation of bound and free radioligand.     

Comparison of 3 AT1 receptor binding assays: filtration assay, ScreenReady Target, and WGA Flashplate

In this article, the study of 3 different angiotensin II type 1 (AT(1)) receptor binding assays in terms of reproducibility, robustness, and feasibility for high-throughput screening (HTS) is described. The following methods were used: a nonhomogeneous filtration assay in a 96-well format using CHO-AT(1) cell membranes and 2 homogeneous assays, which include the commercially available ScreenReady Target for the AT(1) receptor and the wheat germ agglutinin (WGA) Flashplate, which was coated "in-house" with the CHO-AT(1) cell membranes. Receptors were labeled with [(125)I]-Sar(1)-Ile(8)-angiotensin II, and radioligand binding was displaced using the antagonist losartan and the natural agonist angiotensin II. Reproducible K(d), B(max), and K(i) values and good total binding/nonspecific binding (TB/NSB) ratios were obtained with both the ScreenReady Targets and the filtration assay, whereas the WGA Flashplates showed unacceptably high nonspecific binding and high variation when applied as a homogeneous assay. However, when applied as a heterogeneous assay (i.e., when a wash step at the end of the assay is included), the results were significantly better. Interestingly, ligand affinities were consistently lower in Flashplate-based assays than in the filtration assay. This may be due to the immobilization of the receptors onto the solid surface of the plate, affecting their conformation. In terms of reproducibility, robustness, and feasibility for HTS, the authors conclude that the ScreenReady Target plates are most suitable for AT(1) receptor binding screening.     

Glutaraldehyde pretreatment blocks phospholipase A2 modulation of adrenergic receptors

Treatment of rat cerebral cortical membranes with phospholipase A2 affects, in a parallel fashion, beta-, alpha 1- and alpha 2-adrenergic receptor binding, but not the affinity of these receptors for their respective ligands. Pretreatment of membranes with 0.1 percent glutaraldehyde blocks the effects of phospholipase A2 on adrenergic receptor binding. The results support the hypothesis that desensitization or "masking" of adrenergic receptors may involve changes in membrane lipid composition. Furthermore, glutaraldehyde may prove a useful tool in the investigation of the dynamic roles of lipids in receptor function and more specifically, their regulation and coupling to physiological events.     

Regulation of alpha- and beta-adrenergic receptor subclasses by gonadal steroids in human myometrium

Both alpha- and beta-adrenergic receptors have been identified in the human myometrium by radioligand binding. Both adrenergic receptor subclasses have been shown to mediate the contractile response of the uterus upon catecholamine stimulation: alpha-adrenergic receptors cause uterine contraction while beta-adrenergic receptors induce relaxation. We have identified alpha 1- and alpha 2-adrenergic receptors in myometrial membranes using the newly developed radiolabelled specific antagonists [3H]-prazosin and [3H]-rauwolscine. This enabled us to characterize both receptor subclasses individually. Beta adrenergic receptors were identified using the radiolabelled antagonist (-)-[3H]-dihydroalprenolol. Binding of radioligands to the myometrial membrane receptors was rapid, readily reversible, of high affinity and stereoselective. The total number of alpha 1-, alpha 2- and beta-receptors was determined by Scatchard analysis of radioligand saturation binding and the beta/beta 2-receptor ratio was determined by computer analysis of the beta 2-selective antagonist ICI 118 551) (-)-[3H]-dihydroalprenolol competition binding curves. This enabled us to study the regulation of both alpha- and beta-receptor subclasses under various physiological and pharmacological conditions in the human, i.e., during different phases of the menstrual cycle, in postmenopausal women and during depo-progestin (Medroxyprogesterone acetate) therapy. Only the alpha 2- and beta 1-adrenergic receptor concentrations were found to be subjected to gonadal steroid regulation. The number of alpha 2- and beta 1-adrenergic receptors increased concomitantly with circulating plasma oestradiol levels. This effect was counteracted by progesterone. The number of alpha 1- and beta 2-adrenergic receptors was unaffected by the gonadal steroid environment. These results are an example of the heteroregulation of membrane receptors by oestrogens and progesterone and cast new light on the regulatory mechanisms involved in uterine contractility in the human.     

Human cardiac beta-adrenergic receptors: subtype heterogeneity delineated by direct radioligand binding

Human myocardial beta-adrenergic receptors were directly identified and characterized using the high affinity antagonist radioligand [125I]iodocyanopindolol. Beta 1 and beta 2 adrenergic receptors were found to coexist in both the left ventricle and right atrium. The relative proportions of the two receptor subtypes were determined by the use of competition radioligand binding and computer modelling techniques employing the subtype selective agents atenolol (beta 1 selective) and zinterol (beta 2 selective). The left ventricle contains 86 +/- 1% beta 1 and 14 +/- 1% beta 2 adrenergic receptors while the right atrium contains 74 +/- 6% beta 1 and 26 +/- 6% beta 2 adrenergic receptors. The direct demonstration of beta 2 adrenergic receptors in the human heart, with a higher proportion in the right atrium agrees with pharmacologic data and supports the notion that chronotropic effects of adrenergic agonists in man may be mediated by both beta 1 and beta 2 adrenergic receptors.     

Glycerol, sodium phosphate, and sodium chloride permit the solubilization and partial purification of rat hepatic alpha 1-receptors by 3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate

CHAPS [3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate], a zwitterionic detergent, has been used to solubilize the rat hepatic alpha 1-adrenergic receptor. Although the use of this detergent alone permitted a poor receptor solubilization, the inclusion of sodium phosphate, sodium chloride, and glycerol to the medium allowed 30% of the binding activity observed in plasma membranes to be recovered. Binding of the selective alpha 1-adrenergic antagonist, [3H]prazosin, by the solubilized preparation was saturable and of high affinity. In addition, binding of the radioligand was inhibited by a variety of adrenergic agents with affinity, specificity, and stereoselectivity comparable to that observed in plasma membranes. The use of glycerol in the solubilization medium permitted recovery of the solubilized receptor in a stable form (T1/2 = 72 h at 4 degrees C). Sequential affinity and size-exclusion gel chromatography allowed a 1000-fold purification of the solubilized receptor. The Stokes' radius and the apparent molecular mass of the purified receptor-Chaps complex (48.4 A and 160,000 Da, respectively), determined by gel filtration chromatography, were similar to those previously obtained for the rat hepatic alpha 1-receptor purified after solubilization with the nonionic detergent digitonin. These data indicate that the combination of Chaps, sodium phosphate, sodium chloride, and glycerol permitted the solubilization and partial purification of hepatic alpha 1-receptor in an active and stable form. The use of this technique might be useful for the solubilization of other membrane-bound proteins by Chaps whose biophysical characteristics make it an ideal detergent for reconstitution experiments.     

Sulfhydryl group(s) in the ligand binding site of the D-1 dopamine receptor: specific protection by agonist and antagonist

An iodinated compound, [125I]-8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin -7-ol, has been recently reported [Sidhu, A., & Kebabian, J.W. (1985) Eur. J. Pharmacol. 113, 437-440] to be a specific ligand for the D-1 dopamine receptor. Due to its high affinity and specific activity, this ligand was chosen for the biochemical characterization of the D-1 receptor. Alkylation of particulate fractions of rat caudate nucleus by N-ethylmaleimide (NEM) caused an inactivation of the D-1 receptor, as measured by diminished binding of the radioligand to the receptor. The inactivation of the receptor sites by NEM was rapid and irreversible, resulting in a 70% net loss of binding sites. On the basis of Scatchard analysis of binding to NEM-treated tissue, the loss in binding sites was due to a net decrease in the receptor number with a 2-fold decrease in the affinity of the receptor for the radioligand. Receptor occupancy by either a D-1 specific agonist or antagonist protected the ligand binding sites from NEM-mediated inactivation. NEM treatment of the receptor in the absence or presence of protective compound abolished the agonist high-affinity state of the receptor as well as membrane adenylate cyclase activity. The above-treated striatal membranes were fused with HeLa membranes and assayed for dopamine-stimulated adenylate cyclase activity. When the sources of D-1 receptors were from agonist-protected membranes, the receptors retained their ability to functionally couple to the HeLa adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific Binding of [11C]Spiroperidol in Rat Brain In Vivo

Spiroperidol labeled with carbon-11, a short-lived positron-emitting radionuclide, was used to determine the time course of specific binding of this radioligand to the neuroleptic receptor in vivo in the rat. The three major bran pools--specifically bound, nonspecifically bound, and free (unbound)--were determined over a 60-mm time course by a rapid filtration technique, utilizing (-)- and (+)-butaclamol pretreatments to assess total and nonspecifically bound activities, respectively, in striatum and cerebellum. The ratio of specifically to nonspecifically bound pools in the striatum was 4.1 at 30 min and 5.1 at 60 min. Thus [11C]spiroperidol may be useful for labeling neuroleptic receptors in vivo for serial studies using positron emission transaxial tomography.     

Solubilization and hydrophobic immobilization assay of a cAMP binding protein from Dictyostelium discoideum plasma membranes

A cAMP binding site present on isolated plasma membranes of aggregation-competent $\text{D.}\text{discoideum}$ cells has been solubilized with the nonionic detergent Emulphogene BC-720. An assay has been developed based on the principle of hydrophobic chromatography, in which the detergent solubilized cAMP binding protein is immobilized on alkyl-agarose beads at low detergent concentration. This allows the necessary rapid separation of bound and free [³H]-cAMP by filtration of the beads. The kinetics and nucleotide specificity of the detergent solubilized cAMP binding protein are comparable to those of the cAMP chemotactic receptor on intact cells and plasma membranes. The alkyl-agarose bead assay may have general utility for the assay of detergent solubilized membrane receptors.     

Preponderance of alpha 2- over beta 1-adrenergic receptor sites in human fat cells is not predictive of the lipolytic effect of physiological catecholamines

Adrenergic control of human fat cell lipolysis is mediated by two kinds of receptor sites that are simultaneously stimulated by physiological amines. To establish a correlation between the binding characteristics of the receptor and biological functions, the ability of physiological amines to stimulate or inhibit isolated fat cell lipolysis in vitro was compared to the beta- and alpha 2-adrenoceptor properties of the same fat cell batch. The beta-selective antagonist (-)[3H]dihydroalprenolol ([3H]DHA) and the alpha 2-selective antagonists [3H]yohimbine ([3H]YOH) and [3H]rauwolscine ([3H]RAU) were used to identify and characterize the two receptor sites. Binding of each ligand was rapid, saturable, and specific. The results demonstrate 1) the weaker lipolytic effect of epinephrine compared with norepinephrine. This can be explained by the equipotency of the amines at the beta 1-sites and the higher affinity of epinephrine for alpha 2-adrenergic receptors. 2) The preponderance of alpha 2-adrenergic receptor sites labeled by [3H]YOH (Bmax, 586 +/- 95 fmol/mg protein; KD, 2.7 +/- 0.2 nM) or [3H]RAU (Bmax, 580 +/- 100 fmol/mg protein; KD, 3.7 +/- 0.1 nM). These two ligands can be successfully used to label alpha 2-adrenergic receptor sites. 3) The beta 1-adrenergic receptor population labeled by [3H]DHA(Bmax, 234 +/- 37 fmol/mg protein; KD, 1.8 +/- 0.4 nM), although a third as numerous as the alpha 2-adrenergic population, is responsible for the lipolytic effect of physiological amines and is weakly counteracted by simultaneous alpha 2-adrenergic receptor stimulation under our experimental conditions. It is concluded that, in human fat cells, the characterization of beta 1- and alpha 2-adrenergic receptors by saturation studies or kinetic analysis to determine affinity (KD) and maximal number of binding sites (Bmax) is not sufficient for an accurate characterization of the functional adrenergic receptors involved in the observed biological effect.     

Comparison of muscarinic and beta adrenergic receptors between bovine peripheral lung and tracheal smooth muscles: a striking difference in the receptor concentration

This study was undertaken to compare the activity of muscarinic and beta adrenergic receptors in bovine peripheral lung to the corresponding receptor activity in tracheal smooth muscle. We used [3H] quinuclidinyl benzilate (QNB) and [3H]dihydroalprenolol (DHA) to measure muscarinic and beta receptor activity, respectively. Binding to QNB and DHA at 25 degrees C was rapid, reversible, saturable and of high affinity. The order of potency for cholinergic and adrenergic agents competing for binding was compatible with muscarinic and beta 2 adrenergic potencies. We found that the concentration of muscarinic receptor binding sites was 37-fold greater in the tracheal muscle preparation (2805 +/- 309 fmol/mg protein) than in the peripheral lung preparation (76 +/- 28 fmol/mg protein). Unlike muscarinic receptors, the lung contained 8-fold higher concentration of the beta adrenergic receptors than did the tracheal muscle (1588 +/- 417 vs. 199 +/- 42 fmol/mg protein). The dissociation constant or the agonist's inhibitory constant (Ki) for either receptor binding site, however, was not significantly different between the two tissues. Furthermore, in vitro contraction studies showed that the response of tracheal muscle strips to methacholine was markedly greater than the response of peripheral lung strips, a finding consistent with the QNB binding result. The muscle but not the peripheral lung strip exhibited a relaxing response to epinephrine. Our data indicate a striking quantitative difference in muscarinic and beta adrenergic receptors between lung tissue and tracheal muscle, and that each receptor in the lung is qualitatively similar to the corresponding receptor in the muscle.