A reliable assay for beta-adrenoceptors in intact isolated human fat cells with a hydrophilic radioligand, [3H]CGP-12177

The beta-adrenergic receptors of isolated human fat cells were identified using a new hydrophilic beta-adrenergic radioligand (+/-)[3H]CGP-12177. The results were compared with those from [3H]dihydroalprenolol binding to fat cells and membranes. [3H]CGP-12177 binding to isolated fat cells showed lower nonspecific binding (less than 15% of total binding) than the lipophilic [3H]dihydroalprenolol (40-60%) at 3 times the KD. At 37 degrees C, [3H]CGP-12177 binding was rapid, reversible, of high affinity (1.2 +/- 0.3 nM) and saturable. The total number of binding sites per cell in subcutaneous adipocytes was 25,000 +/- 6,000 and was equivalent to that found using membrane fractions. Displacement of [3H]CGP-12177 bound to adipocytes by propranolol was stereoselective, consistent with competition at a single site, and had the same characteristics as in membranes. The displacement curves of the beta 1-selective antagonists (atenolol and betaxolol) were biphasic, the high affinity displacement accounting for 70% of the total binding sites. Beta-adrenergic agonists also competed with [3H]CGP-12177 binding in the order of potency: (-) isoproterenol greater than (-) norepinephrine greater than (-) epinephrine, similar to that found in membranes and in in vitro studies on the lipolytic activity of isolated fat cells. This study demonstrates that the sites specifically labeled by [3H]CGP-12177 are the physiological beta-adrenoceptors and also shows that the ligand is better than [3H]dihydroalprenolol for the accurate identification of these receptors in intact human adipocytes. The methodology, which requires biopsies of less than 1 gram of adipose tissue, can be of potential interest for clinical studies investigating the status of fat cell beta-adrenoceptors in various pathophysiological situations.     

Pharmacological evaluation of plasma membrane beta-adrenoceptors in rat hearts using the tissue segment binding method

This study evaluates beta-adrenoceptors in rat atria and ventricle using the tissue segment binding method and compares the results with those obtained using conventional homogenate binding assays. In studies with tissue segment binding, the hydrophilic radioligand [(3)H]-CGP12177 selectively bound to plasma membrane beta-adrenoceptors, and the B(max) levels were significantly higher than those obtained with homogenate binding. However, both binding approaches revealed similar proportions of beta(1)- and beta(2)-adrenoceptors. The regional distribution of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts were also determined using tissue segment binding. Abundance of beta-adrenoceptors and proportion of beta(1)-adrenoceptors were higher in atria than in ventricle, but there was no significant difference between right and left atria or within ventricle (right and left ventricle free walls, apex, and interventricular septum). To establish the ability of the tissue segment binding method to study beta-adrenoceptor regulation such as the internalization of receptors, the effect of prolonged exposure of rat ventricle to (-)-isoprenaline was also investigated by using tissue segments and homogenate binding. Incubation with (-)-isoprenaline for 1 h in vitro caused a concentration-dependent decrease in the density of beta-adrenoceptors, predominantly beta(2)-adrenoceptors, when assessed with tissue segment binding method. In contrast, the subtype-specific change after treatment with (-)-isoprenaline was not detected using homogenate binding. In summary, the tissue segment binding method with [(3)H]-CGP12177 enables a more precise quantitation of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts and is suitable for studying their regulation.     

Direct assessment of beta-adrenergic receptors in intact rat adipocytes by binding of [3H]CGP 12177. Evidence for agonist high-affinity binding complex and for beta 1 and beta 2 receptor subtypes

The characteristics of the binding of the hydrophilic beta-adrenergic antagonist [3H]CGP 12177 to intact rat adipocytes were studied at 37 degrees C and 6 degrees C. At both temperatures and at 90% saturation, the non-specific binding was less than 30% of the total binding. At 37 degrees C, specific [3H]CGP 12177 binding was rapid, reversible of high affinity (1.8 +/- 0.4 nM) and saturable. The number of specific binding sites per adipocyte increased with the fat cell size (about 35 000 and 115 000 sites per cell in adipocytes with diameters of 60 microns and 88 microns, respectively) but remained constant when expressed per unit fat cell surface area. Displacement of [3H]CGP 12177 bound to adipocytes by unselective and selective beta-antagonists was stereospecific, had the same characteristics as those found in adipocyte membranes and showed a heterogeneous specificity for beta 1 and beta 2 adrenergic subtypes. In contrast, beta agonist competition curves, which modeled to two affinity-states of binding, showed high-affinity-state Kd values for agonists 10-25-times higher than those found in membranes under the same experimental conditions. At 6 degrees C, although the number and affinity of the specific binding sites for [3H]CGP 12177 were the same as those found at 37 degrees C, the Kd value for (-)-isoproterenol binding to the high affinity state of these sites (3.0 +/- 0.5 nM) was 25-times lower than at 37 degrees C and similar to the value found in membrane preparations (1.5-4 nM). These results show that the [3H]CGP 12177 specific binding sites detected on intact adipocytes represent the physiological beta-adrenergic receptors. Moreover, this study extends to the adipocyte the validity of the model recently proposed for other cell lines, according to which in intact cells, but not in membranes, agonist-binding promotes a rapid and temperature-dependent conformational change of the beta-adrenergic receptors, leading to a progressive loss of capacity of agonists to form a high-affinity complex.     

Some Unique Properties of CGP-12177

Abstract

CGP-12177, like isoproterenol, has a lower affinity for desensitized receptors. Experiments were performed to study whether this property of CGP-12177 is due to its partial agonist activity or its impermeability for membranes. Reduced binding of [³H]DHA to desensitized receptors at 0°C as well as a reversal of the reduced binding in the presence of digitonin indicate a permeability barrier. A partial agonist effect of CGP-12177 was only found in intact C6 cells and neither in C6 membranes nor in S49 cells. This peculiar effect in intact C6 cells is stereospecific and not due to an inhibition of phosphodiesterases.     

Temperature-dependent binding of hydrophilic beta-adrenergic receptor ligands to intact human lymphocytes

Important differences in binding characteristics between agonists and antagonists of the beta-adrenergic receptor have been described. However, these observations have been complicated since most available antagonists are much more lipophilic than agonists. In order to separate out those binding characteristics of agonist vs. antagonist from those characteristics of lipophilic vs. hydrophilic ligands, we have studied competition of the hydrophilic ligands isoproterenol (agonist) and CGP-12177 (antagonist) with [125I]iodopindolol binding in intact human lymphocytes. Analyzing competition curves from assays performed at 13 degrees C, 25 degrees C and 37 degrees C we demonstrated that at lower temperatures there was a decrease in IC50 for isoproterenol but not for CGP-12177. Using cells preincubated with isoproterenol then extensively washed, competition curves with both isoproterenol and CGP-12177 were biphasic, and characterized by the appearance of a population of receptors with a low affinity for both hydrophilic ligands. Furthermore, at lower temperatures the biphasic nature of these curves was accentuated and was characterized by a 6-fold and 40-fold increase in the apparent KD of a population of low affinity sites for isoproterenol and CGP-12177, respectively.     

Characterization and Regulation of β1-Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Intact human neuroepithelioma SK-N-MC cells bound the beta-adrenergic antagonist (-)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to beta-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol much greater than norepinephrine greater than epinephrine, which is indicative of a beta 1-subtype receptor. Membranes prepared from the cells bound (-)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the beta 1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the beta 2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only beta 1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the beta 1- and beta 2-adrenergic receptors revealed the presence of a very strong beta 1-adrenergic receptor mRNA signal, while under the same conditions no beta 2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of beta 1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.     

Effect of the Tricyclic Antidepressant Desipramine on β-Adrenergic Receptors in Cultured Rat Glioma C6 Cells

Rat glioma C6 cells, cultured in the presence of the tricyclic antidepressant desipramine, lost a significant number of beta-adrenergic receptors in a time- and dose-dependent manner. A similar loss was observed whether binding was determined on intact cells with the hydrophilic beta-adrenergic antagonist (+/-)-[3H]4-(3-tert-butylamino-2-hydroxypropoxyl)benzimidazole-2-o n HCl ([3H]CGP-12177) or on cell lysates with the more hydrophobic antagonists [125I]iodocyanopindolol or [3H]dihydroalprenolol. When stimulated with the agonist isoproterenol, desipramine-treated cells accumulated less cyclic AMP than control cells. The affinity of the beta-adrenergic receptors for either antagonist or agonist was unchanged after desipramine treatment. Desipramine interacted only weakly with the receptors and competed for [125I]iodocyanopindolol binding with a Ki of 30 microM. The presence in the culture medium of alprenolol or propranolol, potent beta-adrenergic antagonists, however, did not prevent the reduction in receptors by desipramine. Desipramine also caused a loss of beta-adrenergic receptors from cells maintained in serum-free medium and the cells themselves did not contain or secrete endogenous catecholamines. Although desipramine is a potent inhibitor of catecholamine uptake, it appears unlikely that the observed loss of beta-adrenergic receptors in rat glioma C6 cells exposed to the drug is due to an increase in extracellular catecholamine levels or to a direct interaction with the receptors.     

The effect of hydrogen peroxide upon beta-adrenoceptor density and function in C6 rat glioma cells

Hydrogen peroxide has been suggested to play an important role in the pathogenesis of Alzheimer's disease. In the present study, the effects of hydrogen peroxide upon the functional integrity of beta-adrenoceptors have been investigated in C6 glioma cells. Treatment of cells for 24 h with hydrogen peroxide in serum-free medium produced a concentration-dependent cell toxicity, seen both using cell counting and LDH release into medium as end point. There were no large nor consistent changes in either the density of cell surface beta 1, or beta 2-adrenoceptors, measured using the hydrophilic ligand [3H](-)-CGP 12177, nor in either basal, forskolin and isoprenaline-stimulated cAMP responses, following hydrogen peroxide treatment. It is concluded that the decreased adenylyl cyclase activity and responsiveness to Gs stimulation found in post-mortem brain samples from Alzheimer's disease autopsy cases is unlikely to be mediated by hydrogen peroxide.     

Internalization-sequestration and degradation of cholecystokinin (CCK) in tumoral rat pancreatic AR 4-2 J cells

Cholecystokinin (CCK) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas, after preincubation with 20 nM dexamethasone. At steady state binding at 37 degrees C (i.e., after a 5 min incubation), less than 10% of the radioactivity of [125I]BH-CCK-9 (3-(4-hydroxy-[125I]iodophenyl)propionyl (Thr34, Nle37) CCK(31-39)) could be washed away from intact cells with an ice-cold acidic medium, suggesting high and rapid internalization-sequestration of tracer. By contrast, more than 85% of the tracer dissociated rapidly after a similar acid wash from cell membranes prelabelled at steady state. In intact AR 4-2 J cells, internalization required neither energy nor the cytoskeleton framework. Tracer internalization was reversed partly but rapidly at 37 degrees C but slowly at 4 degrees C. In addition, two degradation pathways of the tracer were demonstrated, one intracellular and one extracellular. Intracellular degradation occurred at 37 degrees C but not at 20 degrees C and resulted in progressive intracellular accumulation of [125I]BH-Arg that corresponded, after 1 h at 37 degrees C, to 35% of the radioactivity specifically bound. This phenomenon was not inhibited by serine proteinase inhibitors and modestly only by monensin and chloroquine. Besides, tracer degradation at the external cell surface was still observable at 20 degrees C and yielded a peptide (probably [125I]BH-Arg-Asp-Tyr(SO3H)-Thr-Gly). This degradation pathway was partly inhibited by bacitracin and phosphoramidon while thiorphan, an inhibitor of endopeptidase EC 3.4.24.11, was without effect.     

Dependence of kinetic constants of intact human lymphocyte beta2-adrenoreceptors on the nature of the radioligands used

An analysis of human peripheral blood intact mononuclear lymphocyte beta 2-adrenoreceptors showed that the hydrophilic radioligand 3H-CGP-12177 binds at various temperatures only to surface receptors. The density of receptors as determined by the binding of the lipophilic antagonist 125I-CYP at 37 degrees C is 2 times as high as that at 4 degrees C. The affinity of lymphocyte beta 2-adrenoceptors for 1-isoproterenol measured by the labeled ligand displacement at low (4 degrees C) temperatures is by two or three orders of magnitude higher than that at 37 degrees C. The thiol-alkylating agent, N-ethylmaleimide (NEM) causes oppositely directed changes in the density of beta 2-adrenoceptors on intact lymphocytes depending on their original density. NEM increases the affinity of beta 2-adrenoreceptors for the hormone (1.5-12-fold). The state and regulation of human peripheral blood lymphocyte beta 2-adrenoreceptors depend on the hormonal status of patients at the moment of blood sample collection.     

Characterization of beta-adrenoceptors responsible for venom production in the venom gland of the snake Bothrops jararaca

We have shown that the stimulation of beta-adrenoceptors is an important step in venom production in the Bothrops jararaca venom gland. In the present study, the pharmacological profile of the beta-adrenoceptor present in Bothrops jararaca venom gland was characterized by radioligand binding assay and by the ability of isoprenaline to promote accumulation of cyclic AMP in dispersed secretory cells. In both cases, the venom glands were obtained from non-extracted snakes (quiescent stage) or from snakes which venom was extracted 4 days before sacrifice (venom production stimulated stage). [125I]-iodocyanopindolol ([125I]-ICYP) bound to extracted gland membranes in a concentration-dependent and saturable manner, but with low affinity. Propranolol, beta1- or beta2-selective adrenoceptors ligands displaced the [125I]-ICYP binding with low affinity, while selective beta3-adrenoceptor ligands did not displace the [125I]-ICYP binding. The displacement of [125I]-ICYP by propranolol was similar in non-extracted and extracted glands, showing the presence of beta-adrenoceptors in both stages. In dispersed secretory cells of non-extracted glands, isoprenaline (1 microM) increased the cyclic AMP production and propranolol (10 microM) was able to block this effect. On the other hand, in extracted glands, isoprenaline had no effect. The results suggest that the beta-adrenoceptors present in the Bothrops jararaca venom glands are different from those (beta1, beta2 or beta3) described in mammals, but are coupled to the Gs protein, like the known beta-adrenoceptor subtypes. Moreover, previous in vivo stimulation of venom production desensitizes the beta-adrenoceptors system and, although the receptors could be detected by binding studies, they are not coupled to the Gs protein, indicating that beta-adrenoceptors stimulation contributes to the initial steps of venom synthesis.     

Different changes of plasma membrane beta-adrenoceptors in rat heart after chronic administration of propranolol, atenolol and bevantolol

Recently, tissue segment binding method with a hydrophilic radioligand [(3)H]-CGP12177 was developed to detect plasma membrane beta-adrenoceptors in rat heart (Horinouchi et al., 2006). In the present study, propranolol (40 mg kg(-1) day(-1)), atenolol (40 mg kg(-1) day(-1)) and bevantolol (200 mg kg(-1) day(-1)) were administered to rats for 6 weeks, and the changes of plasma membrane beta-adrenoceptors and their mRNA expression in rat ventricle were examined. Chronic administration of propranolol increased the beta(1)-adrenoceptors but decreased the beta(2)-adrenoceptors without changing total amount of plasma membrane beta-adrenoceptors. Atenolol increased both plasma membrane beta(1)- and beta(2)-adrenoceptors, whereas bevantolol had no effect on the beta-adrenoceptor density and their subtype proportions. In contrast, the density of beta-adrenoceptors detected in conventional homogenate binding study was extremely low (approximately 60% of plasma membrane beta-adrenoceptors detected with the tissue segment binding method) and the effects of chronic administration of beta-adrenoceptor antagonists were not necessarily in accord with those at the plasma membrane beta-adrenoceptors. The mRNA levels of beta(1)- and beta(2)-adrenoceptors were not altered by propranolol treatment, while beta(1)-adrenoceptor mRNA significantly decreased after administration of atenolol or bevantolol without changing the level of beta(2)-adrenoceptor mRNA. The present binding study with intact tissue segments clearly shows that the plasma membrane beta(1)- and beta(2)-adrenoceptors of rat heart, in contrast to the homogenate binding sites and the mRNA levels, are differently affected by chronic treatment with three beta-adrenoceptor antagonists; up- and down-regulations of beta(1)- and beta(2)-adrenoceptors, respectively, by propranolol, and up-regulation of both the subtypes by atenolol, but no significant change in both the subtypes by bevantolol.     

Rapid and reversible disappearance of beta-adrenergic cell surface receptors

The water soluble beta-adrenergic ligand [3H]CGP-12177 was used to measure the cell surface receptors in intact cells. In two cell lines, C6 glioma and WEHI 7 lymphoma cells, -50% of the cell surface receptors disappear within minutes of incubation of the cells with isoproterenol. The receptors can still be detected in homogenates and reappear on the cell surface when cells are washed and reincubated at 37 degrees C. The data agree with a disappearance of the receptors from the cell surface by an agonist-mediated endocytosis.     

Molecular characteristics and evidence for internalization of vasoactive-intestinal-peptide (VIP) receptors in the tumoral rat-pancreatic acinar cell line AR 4-2 J

1. Vasoactive intestinal peptide (VIP) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas [125I]Iodo-VIP binding to cell membranes showed the following IC50 values for unlabeled peptides: VIP, 0.3 nM; peptide His-IleNH2, 2 nM; helodermin, 30 nM; secretin, 100 nM. After incubation with 20 nM dexamethasone, the binding capacity increased twofold but affinities were unchanged. External [125I]iodo-VIP binding to intact cells reached steady state after 5 min at 37 degrees C, while the sequestration-internalization of the [125I]iodo-VIP-receptor complex (tested by cold acid washing) increased progressively, reaching 75% of total binding after 1 h. This phenomenon was blocked at 4 degrees C. Further data with dexamethasone, tunicamycin, cycloheximide, low temperature, and/or phenylarsine oxide, suggested a half-life of 2 days for VIP receptors and the necessity of N-glycosylation for proper translocation. 2. For chemical [125I]iodo-VIP cross-linking bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone gave the best yield when compared with five other bifunctional reagents. In membranes, the main specifically cross-linked peptide had Mr 66,000 under nonreducing conditions, and migrated with lower velocity (-5%) under reducing conditions. Cross-linking was suppressed by VIP, peptide His-IleNH2 and helodermin (competitively) and also by GTP. In intact cells, the Mr of [125I]iodo-VIP-cross-linked peptides depended on the mode of cell solubilization. After direct solubilization, the major cross-linked radioactivity migrated as a smear of Mr 130,000-180,000 but an Mr-66,000 peptide was also detectable. In contrast, the solubilization of cross-linked cells detached by mild trypsinisation gave mainly the Mr-66,000 labeled peptide. This suggests that most VIP receptors in intact, attached cells were in a high-Mr complex and that mild cell treatment was sufficient to disrupt this complex.     

Desensitization of the beta-adrenergic receptor-coupled adenylate cyclase in cultured mammalian cells. Receptor sequestration versus receptor function

Human A431 and rat glioma C6 cells exposed to isoproterenol underwent a time- and dose-dependent loss of isoproterenol-stimulated adenylate cyclase activity. Desensitization was accompanied by sequestration of beta-adrenergic receptors, which became less accessible to the hydrophilic antagonist 3H-labeled 4-(3-tert-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride ([3H]CGP-12177) and redistributed from the heavier density plasma membrane fraction to a lighter density membrane fraction. Prior treatment of the cells with concanavalin A or phenylarsine oxide blocked sequestration of the receptors but not desensitization of the agonist-stimulated adenylate cyclase. The membranes from such pretreated cells were exposed to alkali to inactivate adenylate cyclase, and the receptors were transferred to a foreign adenylate cyclase by membrane fusion with polyethylene glycol. beta receptors from desensitized cells exhibited a reduced ability to maximally stimulate the foreign adenylate cyclase, but remained accessible to [3H]CGP-12177 in the fused membranes. When isoproterenol-treated cells were washed free of agonist, there was a time-dependent recovery of agonist responsiveness and [3H]CGP-12177-binding sites. Using the fusion technique, the receptors recovered their functional activity in the resensitized cells. In concanavalin A-treated cells, desensitization and resensitization appeared to occur in the absence of receptor sequestration. Finally, membranes from desensitized cells pretreated with concanavalin A were fused with polyethylene glycol and assayed for agonist-stimulated adenylate cyclase. There was no reversal of the desensitized state. Thus, the primary, essential step in the desensitization process is a reduction in functional activity of the beta-adrenergic receptor. In contrast, sequestration of the receptors is not a prerequisite, but a secondary event during desensitization.     

Properties of beta-adrenergic receptors of cultured mammalian cells. Interaction of receptors with a guanine nucleotide-binding protein in membranes prepared from L6 myoblasts and from wild-type and cyc- S49 lymphoma cells

The radiolabeled agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) and antagonist [125I]iodopindolol ([125I]IPIN) were used to investigate the properties of beta-adrenergic receptors on membranes prepared from L6 myoblasts and S49 lymphoma cells. The high affinity binding of (-)-[3H]HBI to membranes prepared from L6 myoblasts was stereoselectively inhibited by the active isomers of isoproterenol and propranolol. The density of receptors determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. The binding of (-)-[3H]HBI was inhibited by guanine nucleotides, suggesting an agonist-mediated association of the receptor with a guanine nucleotide-binding protein, presumably the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase. Results obtained in studies with membranes prepared from wild-type S49 lymphoma cells and the adenylate cyclase-deficient variant (cyc-) were similar to those obtained in experiments carried out with membranes prepared from L6 myoblasts. Thus, the high affinity binding of (-)-[3H]HBI to membranes prepared from wild-type and cyc- S49 lymphoma cells was stereoselectively inhibited by the active isomers of isoproterenol and propranolol, and was inhibited by GTP. Moreover, the density of sites determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. These results suggest either that cyc- cells contain a partially functional Ns, or alternatively, that the inhibitory guanine nucleotide-binding protein (Ni) is capable of interacting with beta-adrenergic receptors.     

Synthesis of the nanomolar photoaffinity GABA(B) receptor ligand CGP 71872 reveals diversity in the tissue distribution of GABA(B) receptor forms

A radioiodinated probe, [125I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA(B) receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain revealed two predominant ligand binding species at approximately 130 and approximately 100 kDa believed to represent the long (GABA(B)R1a) and short (GABA(B)R1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABA(B) receptor-specific antibody confirming the receptor specificity of the photoaffinity probe. Most convincingly, [125I]-CGP 71872 binding was competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, (-)-baclofen, (+)-baclofen and (L)-glutamic acid with a rank order and stereospecificity characteristic of the GABA(B) receptor. Photoaffinity labeling experiments revealed that the recombinant GABA(B)R2 receptor does not bind [125I]-CGP 71872, providing surprising and direct evidence that CGP 71872 is a GABA(B)R1 selective antagonist. Photoaffinity labeling experiments using rat tissues showed that both GABA(B)R1a and GABA(B)R1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABA(B)R1b receptor form was detected in kidney and liver whereas the long GABA(B)R1a form was selectively expressed in the adrenal gland, pituitary, spleen and prostate. We report herein the synthesis and biochemical characterization of the nanomolar affinity [125I]-CGP 71872 and CGP 71872 GABA(B)R1 ligands, and differential tissue expression of the long GABA(B)R1a and short GABA(B)R1b receptor forms in rat and dog.     

Effect of prolonged 5-hydroxytryptamine uptake inhibition by paroxetine on cortical beta 1 and beta 2-adrenoceptors in rat brain

The effects of prolonged (21 day) oral administration of the antidepressants paroxetine (0.9 to 8.9 mg/kg/day) and amitriptyline (2.7 to 27 mg/kg/day), on rat brain cortical beta 1- and beta 2-adrenoceptor numbers and affinities were investigated using [3H]-CGP 12177. Although amitriptyline, 27 mg/kg, caused a significant (p less than 0.05) 20% reduction in the number of beta 1-adrenoceptors, paroxetine, at doses up to 8.9 mg/kg p.o., did not influence binding of [3H]-CGP 12177 to cortical beta 1- or beta 2-adrenoceptors. This study with paroxetine provides further evidence that the down-regulation of central beta 1-adrenoceptors in rat brain after repeated administration is not a property of all antidepressant drugs.     

Human fat cell beta-adrenergic receptors: beta-agonist-dependent lipolytic responses and characterization of beta-adrenergic binding sites on human fat cell membranes with highly selective beta 1-antagonists

Beta-adrenergic receptors were characterized in human fat cell membranes using 125I-labeled cyanopindolol (125I-labeled CYP) and highly selective beta 1-antagonists. The iodinated radioligand bound saturably and specifically to a single class of high affinity binding sites. The number of binding sites determined with 125I-labeled CYP closely agreed with that determined with two other tritiated radioligands: [3H]dihydroalprenolol and [3H]CGP-12,177. Since 125I-labeled CYP does not discriminate between beta 1- and beta 2-adrenoceptors, the densities of the two receptor subtypes were determined from the competition curves of 125I-labeled CYP by highly selective beta 1-antagonists (bisoprolol, ICI-89,406, CGP-20,712A, and LK-204,545). Moreover, in order to enable correlation with binding data, the regulation of adenylate cyclase activity and of lipolysis was tested with various beta-agonist and antagonist compounds. The results obtained on fat cell membranes from abdominal subcutaneous adipose tissue demonstrated the following. 1) 125I-labeled CYP represents a valuable tool for the quantification and the delineation of beta-receptor subtypes. 2) The presence of sodium ions in binding buffers causes a modification of the affinity of beta-sites for some beta-antagonists. 3) The human fat cell beta adrenergic receptor population defined by nonselective radioligands is composed of two subtypes that can be interpreted in terms of classic beta 1- and beta 2-adrenergic receptor subtypes as assessed by competition studies with highly selective antagonists; beta 2-sites are predominant (60-70% of 125I-labeled CYP sites) in the adipocytes of slightly overweight women. 4) Results support the idea that beta 1- as well as beta 2-adrenergic receptors are coupled with adenylate cyclase and involved in the induction of lipolysis. 5) The results focus on the interest in some beta 2-agonist drugs (zinterol, clenbuterol) as partial inductors of lipolysis, with the lipolytic efficacies of these compounds being well correlated with their efficacies at 125I-labeled CYP sites.     

Salicylic acid alters endothelin-1 binding in intact adult rat ventricular myocytes.

Endothelin receptors ET(A)R and ET(B)R form tight receptor-ligand complexes that complicate our understanding of the physiological, pharmacological, and biochemical properties of these receptors. Although radioligand-binding studies have demonstrated the binding of endothelin-1 (ET-1) to ET(A)R to be essentially irreversible, ET(A)R internalize in a ligand-dependent manner, release ET-1, and then recycle to the cell surface. Salicylic acid (SA) reduces ET-1 binding (IC(50) = 10 mmol/L) to recombinant ET(A)R in isolated membranes by promoting dissociation of [(125)I]ET-1. In the present study, SA (5 mmol SA/L) did not alter [(125I)]ET-1 binding to intact adult rat ventricular myocytes. The lack of effect was not due to internalization of receptor-ligand complexes. However, 100 mmol SA/L significantly reduced [(125)I]ET-1 binding to both intact myocytes and isolated membranes. SA induced the phosphorylation p42/44 extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and an unidentified 40-kDa protein on the activating threonine-glutamic acid-tyrosine (T-E-Y) motif. ERK phosphorylation was reduced by a MAP kinase kinase (MEK) inhibitor, PD98059. Phosphorylation of p40 was reduced by the p38 MAP kinase inhibitor SB203580, but not PD98059. However, inhibition of ERK or p38 MAP kinases did not alter the ability of 100 mmol SA/L to induce dissociation of [125I]ET-1. These results suggest that, in the ventricular myocyte, salicylic acid alters the kinetics of ET-1 binding. The results also suggest an allosteric binding site may be present that modulates the dissociation of ET-1 receptor-ligand complexes in response to an as-of-yet unidentified mediator.