Search

Bronchoconstrictive airway disorders such as asthma are characterized by inflammation and increases in reactive oxygen species (ROS), which produce a highly oxidative environment. β2-adrenergic receptor (β2AR) agonists are a mainstay of clinical therapy for asthma and provide bronchorelaxation upon inhalation. We have previously shown that β2AR agonism generates intracellular ROS, an effect that is required for receptor function, and which post-translationally oxidizes β2AR cysteine thiols to Cys-S-sulfenic acids (Cys-S-OH). Furthermore, highly oxidative environments can irreversibly oxidize Cys-S-OH to Cys-S-sulfinic (Cys-SO 2 H) or S-sulfonic (Cys-SO 3 H) acids, which are incapable of further participating in homeostatic redox reactions (i.e., redox-deficient). The aim of this study was to examine the vitality of β2AR-ROS interplay and the resultant functional consequences of β2AR Cys-redox in the receptors native, oxidized, and redox-deficient states. Here, we show for the first time that β2AR can be oxidized to Cys-S-OH in situ, moreover, using both clonal cells and a human airway epithelial cell line endogenously expressing β2AR, we show that receptor redox state profoundly influences β2AR orthosteric ligand binding and downstream function. Specifically, homeostatic β2AR redox states are vital toward agonist-induced cAMP formation and subsequent CREB and G-protein-dependent ERK1/2 phosphorylation, in addition to β-arrestin-2 recruitment and downstream arrestin-dependent ERK1/2 phosphorylation and internalization. On the contrary, redox-deficient β2AR states exhibit decreased ability to signal via either Gαs or β-arrestin. Together, our results demonstrate a β2AR-ROS redox axis, which if disturbed, interferes with proper receptor function.

Background and Purpose: β(2) -Adrenoceptor agonists are important bronchodilators used for the treatment of chronic obstructive pulmonary disease and asthma. Clinical data on β(2) -adrenoceptor agonists show a range of onset and duration of action. We have investigated whether the receptor binding kinetics of β(2) -adrenoceptor agonists can explain their observed onset of action and duration of effect in the clinic., Experimental Approach: [(3) H]-DHA was used to label β(2) -adrenoceptors expressed in CHO-cell membranes (K(d) of 0.084 nM). Competition kinetic experiments were performed in the presence of unlabelled β(2) agonists at 37°C in HBSS containing GTP. To determine the kinetic parameters, three concentrations (10, 3 and 1 ×K(i) ) of the unlabelled compound were employed against a fixed concentration of [(3) H]-DHA (0.6 nM)., Key Results: The clinically used β(2) -adrenoceptor agonists exhibited a range of association and dissociation rates. The kinetic K(d) and the competition K(i) values of the eight β(2) -adrenoceptor agonists examined were strongly correlated, suggesting that the method had produced accurate k(off) and k(on) rates. The kinetic on-rate was highly correlated with equilibrium binding affinity., Conclusions and Implications: Although the β(2) -adrenoceptor agonists displayed a range of kinetic rate parameters, simulations at relevant drug concentrations suggest that receptor kinetics do not play an important role in determining onset of action in the clinic. In addition, it is unlikely that receptor kinetics exert an important influence on the duration of action of these agonists, as indacaterol (once daily dosing) had a shorter residency time at the receptor than salmeterol (twice daily dosing)., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β(2) adrenergic receptor (β(2)AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.

Sequence analysis of the class A G protein-coupled receptors (GPCRs) reveals that most of the highly conserved sites are located in the transmembrane helices. A second level of conservation exists involving those residues that are conserved as a group characterized by small and/or weakly polar side chains (Ala, Gly, Ser, Cys, Thr). These positions can have group conservation levels of up to 99% across the class A GPCRs and have been implicated in mediating helix-helix interactions in membrane proteins. We have previously shown that mutation of group-conserved residues present on transmembrane helices H2-H4 in the β(2)-adrenergic receptor (β(2)-AR) can influence both receptor expression and function. We now target the group-conserved sites, Gly315(7.42) and Ser319(7.46), on H7 for structure-function analysis. Replacing Ser319(7.46) with smaller amino acids (Ala or Gly) did not influence the ability of the mutant receptors to bind to the antagonist dihydroalprenolol (DHA) but resulted in ~15-20% agonist-independent activity. Replacement of Ser319(7.46) with the larger amino acid leucine lowered the expression of the S319L mutant and its ability to bind DHA. Both the G315A and G315S mutants also exhibited agonist-independent signaling, while the G315L mutant did not show specific binding to DHA. These data indicate that Gly315(7.42) and Ser319(7.46) are stabilizing β(2)-AR in an inactive conformation. We discuss our results in the context of van der Waals interactions of Gly315(7.42) with Trp286(6.48) and hydrogen bonding interactions of Ser319(7.46) with amino acids on H1-H2-H7 and with structural water., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Autophagy is a physiological degradative process key to cell survival during nutrient deprivation, cell differentiation and development. It plays a major role in the turnover of damaged macromolecules and organelles, and it has been involved in the pathogenesis of different cardiovascular diseases. Activation of the adrenergic system is commonly associated with cardiac fibrosis and remodeling, and cardiac fibroblasts are key players in these processes. Whether adrenergic stimulation modulates cardiac fibroblast autophagy remains unexplored. In the present study, we aimed at this question and evaluated the effects of b(2)-adrenergic stimulation upon autophagy. Cultured adult rat cardiac fibroblasts were treated with agonists or antagonists of beta-adrenergic receptors (b-AR), and autophagy was assessed by electron microscopy, GFP-LC3 subcellular distribution, and immunowesternblot of endogenous LC3. The predominant expression of b(2)-ARs was determined and characterized by radioligand binding assays using [(3)H]dihydroalprenolol. Both, isoproterenol and norepinephrine (non-selective b-AR agonists), as well as salbutamol (selective b(2)-AR agonist) increased autophagic flux, and these effects were blocked by propanolol (b-AR antagonist), ICI-118,551 (selective b(2)-AR antagonist), 3-methyladenine but not by atenolol (selective b(1)-AR antagonist). The increase in autophagy was correlated with an enhanced degradation of collagen, and this effect was abrogated by the inhibition of autophagic flux. Overall, our data suggest that b(2)-adrenergic stimulation triggers autophagy in cardiac fibroblasts, and that this response could contribute to reduce the deleterious effects of high adrenergic stimulation upon cardiac fibrosis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

2-Choloroethyl Ethyl Sulfide (CEES) exposure causes inflammatory lung diseases, including acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. This may be associated with oxidative stress, which has been implicated in the desensitization of beta-adrenergic receptors (beta-ARs). The objective of this study was to investigate whether lung injury induced by intratracheal CEES exposure (2 mg/kg body weight) causes desensitization of beta-ARs. The animals were sacrificed after 7 days and lungs were removed. Lung injury was established by measuring the leakage of iodinated-bovine serum albumin ([(125)I]-BSA) into lung tissue. Receptor-binding characteristics were determined by measuring the binding of [(3)H] dihydroalprenolol ([(3)H] DHA) (0.5-24 nM) to membrane fraction in the presence and absence of DLDL-propranolol (10 micro M). Both high- and low-affinity beta-ARs were identified in the lung. Binding capacity was significantly higher in low-affinity site in both control and experimental groups. Although CEES exposure did not change K(D) and B(max) at the high-affinity site, it significantly decreased both K(D) and B(max) at low affinity sites. A 20% decrease in beta(2)-AR mRNA level and a 60% decrease in membrane protein levels were observed in the experimental group. Furthermore, there was significantly less stimulation of adenylate cyclase activity by both cholera toxin and isoproterenol in the experimental group in comparison to the control group. Treatment of lungs with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase (PDE) could not abolish the difference between the control group and the experimental group on the stimulation of the adenylate cyclase activity. Thus, our study indicates that CEES-induced lung injury is associated with desensitization of beta(2)-AR., ((c) 2009 Wiley Periodicals, Inc.)

Endogenous polyamines mediate acute metabolic effects and cardiac hypertrophy associated to beta-adrenoceptor stimulation. The aim of this study is to characterize the role of polyamines on beta-adrenoceptor system mediated responses. To this end, the functional interaction of polyamine modifying drugs on isoproterenol-elicited cardiotonic effect, in isolated left atria of male Wistar rats, and their effects on [(3)H]dihydroalprenolol (DHA) binding on beta-adrenoceptors and on adenylyl cyclase activity of membrane heart were studied. Polyamines interact with beta-adrenoceptors in rat heart, as shown by the displacement of [(3)H]DHA binding. Furthermore, putrescine (but not spermidine or spermine) increased adenylyl cyclase activity, elicited a positive inotropism and increased intracellular cAMP. The putrescine effect on adenylyl cyclase was not antagonized by the beta-adrenoceptors blockers, alprenolol and ICI-118,551, and facilitated the isoproterenol effect. Neither alprenolol, atenolol nor ICI-118,551 antagonized putrescine-elicited positive inotropism. However, the effect was abolished in preparations with desensitized beta-adrenoceptors. alpha-Difluoromethylornithine, an inhibitor of ornithine decarboxylase, antagonized the effect of isoproterenol on inotropism and cAMP increase. In addition, putrescine might elicit effects by mechanisms independent of beta-adrenoceptor system, since in left atria with functional desensitized receptors an interaction with ouabain-elicited cardiotonic effect was observed. These results suggest that putrescine may act as a low affinity agonist on beta-adrenoceptors and modulate acute responses mediated by beta-adrenoceptors. These findings may be of importance in the physiology and in diseases involving cardiac beta-adrenoceptors.

The present study was undertaken to assess cardiac function and characterize beta-adrenoceptor subtypes in hearts of diabetic rats that underwent exercise training (ExT) after the onset of diabetes. Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin. Four weeks after induction, rats were randomly divided into two groups. One group was exercised trained for 3 wk while the other group remained sedentary. At the end of the protocol, cardiac parameters were assessed using M-mode echocardiography. A Millar catheter was also used to assess left ventricular hemodynamics with and without isoproterenol stimulation. beta-Adrenoceptors were assessed using Western blots and [(3)H]dihydroalprenolol binding. After 7 wk of diabetes, heart rate decreased by 21%, fractional shortening by 20%, ejection fraction by 9%, and basal and isoproterenol-induced dP/dt by 35%. beta(1)- and beta(2)-adrenoceptor proteins were reduced by 60% and 40%, respectively, while beta(3)-adrenoceptor protein increased by 125%. Ventricular homogenates from diabetic rats bound 52% less [(3)H]dihydroalprenolol, consistent with reductions in beta(1)- and beta(2)-adrenoceptors. Three weeks of ExT initiated 4 wk after the onset of diabetes minimized cardiac function loss. ExT also blunted loss of beta(1)-adrenoceptor expression. Interestingly, ExT did not prevent diabetes-induced reduction in beta(2)-adrenoceptor or the increase of beta(3)-adrenoceptor expression. ExT also increased [(3)H]dihydroalprenolol binding, consistent with increased beta(1)-adrenoceptor expression. These findings demonstrate for the first time that ExT initiated after the onset of diabetes blunts primarily beta(1)-adrenoceptor expression loss, providing mechanistic insights for exercise-induced improvements in cardiac function.

Androgen-independent prostate cancer cells DU-145 express a number of G protein-coupled receptors, including histamine H1 receptors. There is evidence for the presence of beta-adrenoceptors in the human prostate, and in this work we set out to characterise the expression of beta-adrenoceptors by DU-145 cells, their linking to cyclic AMP (cAMP) formation and the possible modulation by histamine H1 receptors of beta-adrenoceptor function. Saturation [3H]-dihydroalprenolol binding indicated that DU-145 cells express moderate levels of beta-adrenoceptors (22.7+/-2.5 fmol/mg protein), which belong to the beta2-subtype as assessed by inhibition by the antagonists ICI-118,551 and CGP-20712A. Inhibition of [3H]-dihydroalprenolol binding by agonists (noradrenaline, adrenaline and isoproterenol) showed the presence of both high-(53-59%) and low-affinity binding sites. beta-Adrenoceptor stimulation with isoproterenol resulted in robust [3H]-cAMP accumulation (10-30-fold of basal, EC50 142 nM; pEC50 6.85+/-0.05). While not having effect of its own on basal [3H]-cAMP accumulation, histamine significantly augmented the beta2-adrenoceptor-induced response (overall effect 152+/-6% of isoproterenol alone) with EC50 1.35 microM (pEC50 5.87+/-0.06). This effect was independent of extracellular Ca2+, insensitive to antagonists/agonists at H1, H2 or H3/H4 receptors and mimicked by drugs containing an imidazole ring in their chemical structure and by imidazole itself. Taken together, our results show that in DU-145 cells histamine augments beta2-adrenoceptor-induced cAMP independently of the activation of known histamine receptors. The effect may involve other mechanisms such as allosteric modulation of beta2-adrenoceptors by the imidazole moiety of histamine.

We have compared the ability of three radioligands, [(125)I]-cyanopindolol, [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol, to label the three human beta-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [(3)H]-CGP 12,177 had very similar affinity for beta(1)- and beta(2)-adrenoceptors (about 40 pM), [(125)I]-cyanopindolol and [(3)H]-dihydroalprenolol had 4- to 6-fold higher affinity for beta(2)- as compared to beta(1)-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [(125)I]-cyanopindolol at beta(3)-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The beta(3)-adrenoceptor affinity of [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol was so low that it could not be estimated within the tested range of radioligand concentrations (up to 4,000 pM and 30,000 pM for [(3)H]-CGP 12,177 and [(3)H]-dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label beta(3)-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [(125)I]-cyanopindolol appears the least unsuitable to label beta(3)-adrenoceptors. There is a need for high-affinity radioligands which are either selective for beta(3)-adrenoceptors or reasonably non-selective among all three beta-adrenoceptor subtypes.

High-level expression of G-protein-coupled receptors (GPCRs) in functional form is required for structure-function studies. The main goal of the present work was to improve expression levels of beta2-adrenergic receptor (beta2-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster beta2-AR gene suitable for high-level expression in mammalian systems has been accomplished. Transient expression of the gene in COS-1 cells resulted in 18 +/- 3 pmol beta2-AR/mg of membrane protein, as measured by saturation binding assay using the beta2-AR antagonist [3H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of beta2-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type beta2-AR at levels of 220 +/- 40 pmol/mg of membrane protein corresponding to 50 +/- 8 microg/15-cm plate. This level of expression is the highest reported so far for any wild-type GPCR, other than rhodopsin. The yield of functional receptor using the single-step affinity purification is 12 +/- 3 microg/15-cm plate. This level of expression now makes it feasible to pursue structure-function studies using EPR. Furthermore, scale-up of beta2-AR expression using suspension cultures in a bioreactor should now allow production of enough beta2-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.

In the present study, we observed that isoproterenol, a beta-adrenergic receptor (beta-AR) agonist, stimulated rat C6 glioma cell proliferation, while propranolol, a beta-AR blocker, greatly reduced the proliferative effect of TNF-alpha on C6 cells. The gene and protein expressions of both beta1- and beta2-ARs were enhanced in C6 cells after TNF-alpha treatment, and the increase in beta-AR was due to an increased number of binding sites and not due to increase in receptor affinity. We further showed that protein kinase C (PKC) was involved in the TNF-alpha-induced beta-AR expression. Collectively, our results indicate that TNF-alpha-induced proliferation in C6 glioma cells might be via the induction and activation of beta-ARs.

The present study aimed to determine the anatomic distribution and developmental profile of alpha(2) and beta adrenoceptors (AR) in marine teleost brain. Alpha 2 and beta adrenoceptors were studied at different developmental stages by using [(3)H]clonidine and [(3)H]dihydroalprenolol, respectively, by means of in vitro quantitative autoradiography. Furthermore, immunohistochemical localization of the receptor subtypes was performed to determine their cellular distribution. Saturation studies determined a high-affinity component of [(3)H]clonidine and [(3)H]dihydroalprenolol binding sites. High levels of both receptors were found in preglomerular complex, ventral hypothalamus, and lateral torus. Dorsal hypothalamus and isthmus included high levels of alpha(2) AR, whereas pretectum and molecular and proliferative zone of cerebellum were specifically characterized by high densities of beta AR. From the first year of life, adult levels of both AR were found in most medial telencephalic, hypothalamic, and posterior tegmental areas. Decreases in both receptors densities with age were prominent in ventral and posterior telencephalic, pretectal, ventral thalamic, hypothalamic, and tegmental brain regions. Immunohistochemical data were well correlated with autoradiography and demonstrated the presence of alpha(2A), alpha(2C), beta(1), and beta(2) AR subtype-like immunoreactivity. Both the neuronal (perikaryal or dendritic) and the glial localization of receptors was revealed. The localization and age-dependent alterations in alpha(2) and beta AR were parallel to plasticity mechanisms, such as cell proliferation in periventricular thalamus, hypothalamus, and cerebellum. In addition, the biochemical characteristics, distribution pattern, and neuronal or glial specificity of the receptors in teleost brain support a similar profile of noradrenergic transmission in vertebrate brain evolution., ((c) 2005 Wiley-Liss, Inc.)

Characteristics of distribution of cholinergic and adrenoceptor structures along the sinoatrial node artery in rat heart were evaluated by autoradiography on semithin sections by determining the density of (3)H-dihydroalprenolol and (3)H-quinuclidinyl benzilate binding sites. The relative density of binding sites for (3)H-dihydroalprenolol and (3)H-quinuclidinyl benzilate was minimum in the functional nucleus of the sinoatrial node and asymmetrically increased to maximum values to cranial (sharply) and caudal (smoothly) directions. The relative level of binding for (3)H-dihydroalprenolol in the perinodal atrial myocardium tissue was markedly lower than in the periarterial zone of the central part of the sinoatrial node and comparable to that for (3)H-quinuclidinyl benzilate.

Arachidonic acid (AA), released in response to muscarinic acetylcholine receptor (mAChR) stimulation, previously has been reported to function as a reversible feedback inhibitor of the mAChR. To determine if the effects of AA on binding to the mAChR are subtype specific and whether AA inhibits ligand binding to other G protein-coupled receptors (GPCRs), the effects of AA on ligand binding to the mAChR subtypes (M1, M2, M3, M4, and M5) and to the micro-opioid receptor, beta2-adrenergic receptor (beta2-AR), 5-hydroxytryptamine receptor (5-HTR), and nicotinic receptors were examined. AA was found to inhibit ligand binding to all mAChR subtypes, to the beta2-AR, the 5-HTR, and to the micro-opioid receptor. However, AA does not inhibit ligand binding to the nicotinic receptor, even at high concentrations of AA. Thus, AA inhibits several types of GPCRs, with 50% inhibition occurring at 3-25 MuM, whereas the nicotinic receptor, a non-GPCR, remains unaffected. Further research is needed to determine the mechanism by which AA inhibits GPCR function.

Rapid eye movement (REM) sleep deprivation induces a cortical down-regulation of beta-adrenergic receptors. Down-regulation of cortical beta-adrenergic receptors is consistently observed after a number of different chronic antidepressant treatments (drugs and electroconvulsive shock). REM sleep deprivation has an antidepressant effect in humans, and in rats, it decreases immobility in the behavioral despair test, an effect also produced by antidepressant treatments. To verify whether REM sleep deprivation also affects hippocampal beta-adrenergic receptors, we carried out the binding of [3H]-dihydroalprenolol ([3H]-DHA) to hippocampal membranes from rats deprived of REM sleep for 96 h. We also determined the binding of [3H]-DHA to brainstem membranes, a brain region where noradrenergic nuclei are located. Rats were deprived of REM sleep using a water tank with multiple small platforms. [3H-DHA] saturation conditions (concentrations ranging from 0.15 to 6 nM) were obtained in a crude hippocampus and brainstem membrane preparation. Nonspecific binding was determined using DL-propranolol in hippocampus homogenates. In the brainstem homogenates, nonspecific binding was determined in the presence of DL-propranolol or L-isoproterenol. The results obtained showed statistically significant down-regulation of beta-adrenergic receptors in both the hippocampus and the brainstem after REM sleep deprivation. In the hippocampus, there was also a significant decrease in the dissociation constant (KD). In the brainstem, a significant decrease in KD was observed when DL-propranolol was used to determine nonspecific binding. The down-regulation of beta-adrenergic receptors in the hippocampus and brainstem suggests the involvement of these brain areas in the antidepressant effect of REM sleep deprivation.

Human placental syncytiotrophoblast is the main barrier for materno-fetal exchange. Analysis of transplacental transport involves the study of ion channels in both the maternal-facing microvillous membrane (MVM) and the fetal-facing basal membrane (BM). Difficulties in having access to intact placenta with conventional electrophysiological methods favour alternative methodologies, such as isolation and reconstitution of membranes in artificial lipid systems. Pre-eclampsia is a major health problem of human pregnancy. The search for altered physiological processes in pre-eclamptic placentae requires the investigation of events at both the microvillous and basal surfaces. The aim of this study was to obtain reliable syncytiotrophoblast plasma membranes from human normal (N) and pre-eclamptic (PE) pregnancies. We describe a protocol which allows for the simultaneous isolation of MVM and BM. The purity of the membranes isolated was evaluated using enzymatic assays, binding studies, Western blotting and immunohistochemistry. Enrichment of alkaline phosphatase activity for MVM was 17 to 21-fold, with 13-16 per cent protein recovery, for both N and PE. Enrichment of adenylate cyclase activity for BM was 9-fold for N, and enrichment of dihydroalprenolol binding to beta-adrenergic receptors was 12-fold for N and 6-fold for PE, with 14 per cent protein recovery for both N and PE. Cross contamination was low and mitochondrial membrane contamination was negligible. We conclude that MVM and BM isolated from placentae of pre-eclamptic women are similar in enrichment and purity to those of healthy women, thus allowing their use in comparative electrophysiological studies.

Purpose: We investigated the presence and functional role of beta-adrenoceptor subtypes in the bladder base and proximal urethra of the female pig., Materials and Methods: Saturation experiments were done with 7 concentrations (0.25 to 16 nM.) of [(3)H]-dihydroalprenolol (NEN Life Science Products, Boston, Massachusetts). Competition experiments with [(3)H]-dihydroalprenolol were performed with unlabeled antagonists (beta1 selective CGP20712A, beta2 selective ICI118551 and beta3 selective SR59230A). In functional studies concentration-relaxation curves to the beta3-agonist BRL37344 were obtained and antagonist affinities for SR59230A were determined., Results: CGP20712A displaced [(3)H]-dihydroalprenolol with low affinity, suggesting that beta1-adrenoceptors were not present. Displacement with ICI118551 in the bladder base and urethra best fitted a 2-site model with 20% and 28% high affinity sites (beta2), respectively. Displacement experiments with SR59230A in the bladder base demonstrated that 59% of binding sites had high affinity (beta3). In the urethra displacement with SR59230A best fitted a 1-site model but with a pK(i) of 7.2 that was intermediate between that expected for beta2 and beta3-adrenoceptors. In functional studies BRL37344 induced relaxation with pEC50 values of 5.5 and 8, and a maximum relaxation response relative to 30 microM. isoprenaline of 79% and 90% in the bladder base and urethra, respectively. The affinity value of SR59230A for the response to BRL37344 was 7.87 and 7.71 in the bladder base and urethra, respectively, which were intermediate between those of beta2 and beta3-adrenoceptors., Conclusions: Apparently beta3-adrenoceptors are the predominant beta-adrenoceptor subtype present in the lower urinary tract of the pig.

Objective: The aims of this study were (1) to evaluate the usefulness of the new beta2-adrenergic stimulant KUR-1246 as a tocolytic agent and (2) to clarify the mechanisms that underlie the diverse inhibitory effects of beta2-stimulants that are seen in human myometria in vitro., Study Design: The displacement of tritiated ([3H]) (-)CGP 12177 (0.4 nmol/L) by KUR-1246 and other beta2-stimulants was examined with human beta(1)- and beta2-receptors present on membrane fractions. The inhibitory effects of these beta2-stimulants on the term-pregnant human myometrium were compared with the use of isometric tension recording and microelectrode methods. Finally, the relationship between [3H]dihydroaloprenolol binding and the magnitude of the tocolytic effect of isoproterenol was examined., Results: KUR-1246 was approximately 80 times and 7 times more selective for beta2-receptors than isoproterenol and ritodrine, respectively. The inhibitory effect of KUR-1246 was as strong as the inhibitory effect of the conventional beta2-adrenergic stimulants. A wide range of inhibitory effects was observed, even when high concentrations of isoproterenol or KUR-1246 were applied. There was a correlation between the degree to which isoproterenol suppressed contractions and the number of [3H]dihydroaloprenolol binding sites on the membrane in each muscle strip., Conclusion: KUR-1246 should be a very useful beta2-adrenergic stimulant for use as a tocolytic agent because of its high selectivity for the beta2-receptor and its potent inhibitory effect. The diversity of the inhibitory effects that are induced by beta2-stimulants is at least partly due to differences in beta2-receptor density among term-pregnant human myometria.

Molecular interactions of androgens with the plasma membrane may produce rapid cardiovascular effects that cannot be explained by the classic genomic mechanisms. In this sense, 5 alpha- and 5 beta-dihydrotestosterone-induced an acute positive inotropic effect in isolated left atrium of rat, an effect which may be due to cAMP-dependent mechanisms. To prove this, intracellular levels of cAMP, after exposure to androgens in the organ bath, and binding to beta(1)-adrenoceptors were evaluated. After a 4-min exposure, 5 alpha- and 5 beta-dihydrotestosterone increased cAMP levels from 3.83+/-0.61 to 6.15+/-1.1 and 11.18+/-2.4 pmol cAMP/mg of protein, respectively. These increases were inhibited by atenolol and not modified by treatment of the rats with reserpine. The androgen-induced cAMP increase seems to be produced via an extracellular interaction, because positive inotropism and raised levels of cAMP were produced by 5 alpha-dihydrotestosterone conjugated with bovine serum albumin (BSA). In addition, it is independent of beta(1)-adrenoceptor activation, because neither androgen displaced [(3)H]dihydroalprenolol binding. Therefore, the androgens induced a positive inotropic effect via a postsynaptic effect that increases intracellular levels of cAMP. This effect is modulated by transcriptional mechanisms or by a protein with a short half-life.

We investigated the possibility that norepinephrine from the human ovary is released after nerve stimulation and that this neurotransmitter is coupled to a steroidogenic response. Biologically significant levels of both norepinephrine and dopamine were found in human ovarian biopsies. [3H]norepinephrine incorporated in vitro was readily released by electrical stimulation in a Ca2+-dependent process. Ovarian membrane preparations exhibited specific binding sites for the beta-adrenergic antagonist [3H]dihydroalprenolol. Displacement of [3H]dihydroalprenolol with zinterol (a specific beta2-agonist) indicated that 72% of these sites were type beta2-receptors. beta-receptors were also present on granulosa cells. Stimulation of granulosa cells with luteinizing hormone or the beta-agonist isoproterenol increased the release of progesterone after 4 d in culture. These results suggest that the sympathetic nerves present in human ovary are coupled to beta-adrenergic receptors present in endocrine cells and, as in nonprimate mammals, appear to participate in the regulation of ovarian function.

Neurons from the superior cervical ganglia (SCG) innervate the submandibular gland and release noradrenaline during the dark phase of the daily photoperiod. Since in the pineal, another structure innervated by sympathetic neurons, nocturnal activation of the SCG is associated with beta-adrenergic sub- and super-sensitivity rhythms, the possible existence of similar phenomena in the rat submandibular gland was assessed. Wistar female rats, kept on a 14:10 light/dark cycle (light from 06:00 to 20:00 h), were sacrificed at 09:00, 14:00, 20:00, 24:00 and 04:00 h. beta-Adrenoceptors were studied by 3H-dihydroalprenolol binding to membrane preparations. The equilibrium dissociation constant (Kd) did not change as a function of time while significant daily variations in maximal binding values (Bmax) were observed with a peak at 20:00 h. Changes in Bmax correlated with a high response of adenylate cyclase to isoproterenol. In addition, when the response in salivary flow to isoproterenol was measured. a shift to the left (about 1 logarithmic unit) in dose-response curves was observed at 19:00-20:00 has compared to 08:00-09:00 h. These daily variations in isoproterenol responsiveness seem not to depend on the pattern of eating since a 24-h starvation or a nocturnal starvation for 16-18 days did not abolish the morning-evening differences in the salivary flow response to isoproterenol. Rather, the results suggest that the daily variations in isoproterenol response correlate with beta-adrenergic super- and sub-sensitivity phenomena associated with the circadian release of noradrenaline from SCG neurons.

In the present work we studied the interaction of alpha-melanocyte-stimulating hormone (alpha-MSH) and ACTH-(1-24) with beta-adrenergic receptors in hypothalamic membranes from rat brain. Saturation curves for [(3)H]dihydroalprenolol-hydrochloride ([(3)H]DHA) binding in the presence of the peptides revealed a decreased binding capacity (Bmax). The dissociation constant (Kd) was, however, not affected by alpha-MSH or ACTH-(1-24). These data indicate a non competitive interaction between these melanocortin peptides and [(3)H]DHA on beta-adrenergic receptors in hypothalamic membranes.

Stable clones of HEK293 cells expressing either FLAG(TM) epitope-tagged, wild type human beta(1)- and beta(2)-adrenoceptors or C-terminally green fluorescent protein (GFP)-tagged forms of these receptors were established. The binding affinity of [(3)H]-dihydroalprenolol and other ligands was little affected by addition of GFP to the C-terminal of either receptor. Isoprenaline induced the internalisation of both beta(1)-adrenoceptor-GFP and beta(2)-adrenoceptor-GFP and following removal of the agonist both constructs were able to recycle to the cell surface. The extent of internalisation of beta(2)-adrenoceptor-GFP produced by isoprenaline was substantially greater than for beta(1)-adrenoceptor-GFP. C-terminal addition of GFP slowed markedly the rate of internalization of both the beta(1)-adrenoceptor and the beta(2)-adrenoceptor in response to isoprenaline. Sustained exposure to isoprenaline (24 h) produced substantially greater levels of downregulation of native beta(2)-adrenoceptor compared to beta(2)-adrenoceptor-GFP although both were equally effectively removed from the plasma membrane. Sustained exposure to isoprenaline resulted in a large fraction of beta(2)-adrenoceptor-GFP becoming trapped in internal vesicles/lysosomes but not degraded. Even after sustained exposure to isoprenaline a significant fraction of beta(1)-adrenoceptor-GFP remained at the cell surface. These results indicate that although GFP tagging of beta-adrenoceptors can provide qualitative visual patterns of agonist-induced receptor trafficking and regulation in HEK293 cells the quantitative details vary markedly from those obtained with the unmodified receptors.

By using immunocytochemistry we previously reported the absence of beta(2) adrenergic receptors on astrocytes in multiple sclerosis (MS) white matter. Here, we measured beta(1) and beta(2) adrenergic receptor concentrations in postmortem brain sections of six MS patients and six controls by using quantitative autoradiography with [(3)H]dihydroalprenolol. White matter contained no beta(1) adrenergic receptors. In white matter of controls low levels of beta(2) adrenergic receptors were detected. In agreement with the immunohistochemical study, we were unable to detect beta(2) adrenergic receptors in both normal appearing white matter and astrogliotic plaques in MS. Concentrations of beta(1) and beta(2) adrenergic receptors in cerebral cortex were not different between controls and MS patients.

Background: Although tachycardia is commonly present in patients with congestive heart failure, its role in the development of congestive heart failure remains unclear. We studied the effect of rapid electrical stimulation of contraction on beta-adrenergic receptor (beta-AR) signal pathway in cultured cardiomyocytes of neonatal rats., Methods and Results: Contraction of cardiomyocytes was induced by electrical stimulation at 50 V with twice the threshold pulse width. beta-ARs were identified by [(3)H]CGP-12177 and [(3)H]dihydroalprenolol. Electrical stimulation reduced cell-surface but not total beta-AR density; the effect was dependent on pacing frequency (a reduction of 11%, 28%, and 18% in cells paced at 2.5, 3. 0, and 3.3 Hz, respectively). This reduction was apparent at 3 hours, in contrast to reduced beta-AR density after exposure to isoproterenol (ISP) for 1 hour. The fraction and inhibition constant of beta-AR binding agonist with high affinity were not affected by rapid electrical stimulation. In cardiomyocytes paced at 3.0 Hz for 24 hours, the response to ISP decreased compared with unpaced cells, 142% versus 204% of baseline with 1 micromol/L ISP, whereas the responses to forskolin or acetylcholine were not different. Treatment of cardiomyocytes with 2,3-butanedione monoxime (10 mmol/L) or taxol (10 micromol/L) inhibited the rapid pacing-induced reduction in beta-AR density., Conclusions: Our results suggest that contractile activity is involved in regulation of cardiac function by modulating the beta-AR system independently of hemodynamic and neurohormonal factors. This may help to elucidate the role of mechanical stress in the development of heart failure.

The genetically epilepsy-prone rat (GEPR) seizure model is characterized by extensive abnormalities in brain noradrenergic function. Earlier studies had suggested that GEPRs might not regulate adrenoceptors in a normal fashion. The purpose of the present study was to determine if GEPR-9s are capable of up and down regulation of alpha(1)- and beta-adrenoceptors in response to increments or decrements in extracellular norepinephrine. Seizure induction has been shown to increase extracellular norepinephrine. Chronic sound or electroshock-induced seizures caused down regulation of beta-adrenoceptors in frontal cortex and in hippocampus from GEPR-9s. Similarly, chronic daily treatment with the norepinephrine reuptake inhibitor desmethylimipramine produced down regulation of beta-adrenoceptors in frontal cortex and in hippocampus from GEPR-9s. As is the case in neurologically normal animals, chronic electroshock-induced seizure did not cause down regulation of beta-adrenoceptors in 6-hydroxydopamine pretreated GEPR-9s. Chronic electroshock treatment also caused up-regulation of alpha(1)-adrenoceptors in frontal cortex but not in hippocampus. In 6-hydroxydopamine pretreated GEPR-9s, chronic electroshock treatment caused a further up-regulation of alpha(1)-adrenoceptors in frontal cortex but not in hippocampus. Taken together, these results indicate that GEPR-9s are capable of up and down regulation of alpha(1)- and beta-adrenoceptors in a manner that is qualitatively similar to the regulation of these receptors in normal animals. Whether the regulation of brain adrenoceptors is quantitatively different in GEPRs from normal animals remains to be established.

The beta3-selective adrenergic receptor ligand BRL 37344 (BRL) was used to differentiate the presence and functional role of beta-adrenergic receptor (betaAR) subtypes in pig tissues. BRL did not stimulate adenylyl cyclase in membrane preparations or increase lipolysis from pig adipocytes. In contrast to some species, BRL appears to be a poor agonist for the pig betaAR and is not a useful betaAR ligand. Based on displacement of [3H]dihydroalprenolol binding, BRL exhibited a 100-fold selectivity for pig betaAR subtypes in adipose and skeletal muscle membranes. The high affinity site was proposed to be the beta2AR. When used as an antagonist, BRL blockade of the high affinity site did not interfere with isoproterenol-stimulated lipolysis but did inhibit adenylyl cyclase activation. Results indicate that the high affinity betaAR (betaAR) is not linked to lipolysis, possibly due to intracellular compartmentalization. Therefore, betaAR subtypes may have function-specific effects.

Prolonged treatment with the beta(2)-adrenergic agonist clenbuterol (0.25 mg kg(-1) s.c. once daily for 10 days) produced a reduction of the relaxant response and cAMP production mediated by stimulation of beta-adrenoceptors in oestrogen-treated rat uterus. Substantial decreases in the relaxant effect of isoproterenol is observed in uterine rings precontracted with 50 mM KCl from clenbuterol-treated rats. The recovery of the relaxant response was also studied and significant differences were seen between acute and prolonged treatment with clenbuterol (P<0.05 vs control). In contrast the relaxant effect of forskolin and 3-isobutyl-1-methylxanthine was similar in untreated or treated rats. Sodium fluoride also showed a relaxant response which was not affected by the treatment with clenbuterol. The radioligand studies showed a reduction in the number of beta-adrenoreceptors after acute and prolonged treatment with clenbuterol in rat uterus. These results suggest that prolonged treatment with clenbuterol caused a desensitization of the relaxant uterine response through beta(2)-adrenoceptors and also showed differences in the recovery of the relaxant response depending on the duration of treatment., (Copyright 2000 Academic Press.)

beta-Adrenergic agonists are potent relaxing agents of airway smooth muscle; however, they are often incapable of fully reversing agonist-mediated contractions. The present study was designed to quantitate the relationship between beta-adrenoceptor binding, signal transduction, and relaxation in rabbit tracheal smooth muscle (TSM). TSM segments contracted with acetylcholine to 25 to 75% maximal contraction were relaxed with cumulative administration of isoproterenol (ISO). A beta-adrenergic receptor "deficit" was found, such that incomplete relaxation was achieved with full receptor occupancy. Binding studies with [(3)H]dihydroalprenolol demonstrated a beta-adrenoceptor density of 33.1 +/- 8.6 fmol/mg protein in control TSM. Paired studies were performed in TSM from rabbits treated with dexamethasone. Relative to control tissues, dexamethasone-treated TSM displayed twice as much relaxation and cAMP production in response to ISO and twice the beta-adrenoceptor density (82.2 +/- 12.3 fmol/mg protein). Dexamethasone did not affect G(i) function, as assessed by the degree of functional antagonism exerted by acetylcholine on ISO-induced relaxations, or beta-adrenoceptor-G(s) coupling, as reflected in high-affinity beta-agonist binding. Collectively, these results demonstrate that corticosteroid administration exerts parallel potentiating effects on beta-adrenoceptor expression and function in rabbit airway smooth muscle.

The binding and displacement of beta-adrenoceptor blockers, [3H]propranolol ([3H]PRP) and [3H]dihydroalprenolol ([3H]DHA), were studied on isolated rat erythrocytes, their membranes and ghosts; the binding of [3H]DHA and a M-cholinoceptor blocker, [3H]quinuclidinylbenzylate ([3H]QNB), on cerebral cortex membranes. In all experiments, ligand-receptor interactions conformed to a model of two pools of receptors in the same effector system and the binding of two ligand molecules to the receptor. The results were similar for the displacement of [3H]PRP, [3H]DHA and [3H]QNB with propranolol, dihydroalprenolol and quinuclidinyl-benzylate, respectively. The parameters of [3H]PRP to beta-adrenoceptor binding for intact erythrocytes were: Kd1 = 0.74+/-0.07 nM, Kd2 = 14.40+/-0.41 nM, B1 = 24+/-2 unit/cell, B2 = 263+/-5 unit/cell; for ghosts, Kd1 = 0.70+/-0.17 nM, Kd2 = 19.59+/-2.59 nM, B1 = 9+/-1 fmol/mg protein, B2 = 39+/-4 fmol/mg protein. Receptor affinities were similar in erythrocytes and ghosts; on the ghost membrane, the number of receptors was considerably lower (B1 = 2 unit/cell, B2 = 6 unit/cell). The parameters of [3H]QNB to M-cholinoceptor binding of the cerebral cortex membrane were the following: Kd1 = 0.43 nM, Kd2 = 2.83 nM, B1 = 712 fmol/mg, B2 = 677 fmol/mg.

Chronic mild unpredictable stress, which reduces rewarded behaviour in rats, is becoming increasingly popular as an animal model of depression. The effect of chronic mild stress (applied to animals housed five per cage for 15 days) on forced swimming and open field behaviour, and on beta-adrenoceptor binding was studied in naive rats and after the denervation of the locus coeruleus projections by DSP-4 (50 mg kg(-1)) treatment. In the forced swimming test, chronic mild stress reduced the immobility time on the second day of testing in both vehicle- and DSP-4-treated rats, indicating rather an antidepressant-like effect. This antidepressant-like effect of chronic mild stress in the forced swimming test was not present in individually housed rats which suggests that this paradigm is sensitive to housing conditions. Stress had no clear effect on the open field locomotion in naive animals (but caused a reduction in defecations), but completely blocked the DSP-4-induced decrease in the exploratory activity. As measured by 3H-dihydroalprenolol binding, DSP-4 treatment increased the beta-adrenoceptor affinity in the frontal cortex and the number of binding sites in the hippocampus and in the cerebral cortex (total-frontal cortex). Stress had no effect on the beta-adrenoceptor binding in the frontal cortex and cerebral cortex, but prevented the increase in affinity caused by DSP-4 treatment in the frontal cortex. In the hippocampus, chronic mild stress and DSP-4 treatment increased the number of beta-adrenoceptor binding sites. Neither chronic mild stress nor DSP-4 treatment had any effect on CCK(B) receptor binding in the cerebral cortex and striatum. These results show that chronic mild stress applied to group-housed rats can prevent the development of certain behavioural and biochemical changes caused by the denervation of the locus coeruleus projection areas.

It is now known that rapid placental and fetal development is associated with elevated levels of circulating high density lipoprotein (HDL) in pregnant women. The main structure implicated in the maternal-fetal exchange is the syncytiotrophoblast, composed of a brush border membrane (BBM), facing the mother, and a basal plasma membrane (BPM), facing the fetus. In order to understand the mechanisms controlling the placental and fetal supplies of cholesterol, we purified both BBM and BPM and verified the presence of HDL binding sites in these membranes. Binding studies using(125)I-HDL(3)show a single affinity binding site on BPM which has a dissociation constant (K(d)) of 3.45+/-0.43 microg protein/ml and a maximal binding capacity (B(max)) of 5.46+/-1.69 microg protein/mg membrane proteins. In BBM, we observed two affinity binding sites, one with a K(d)of 0.62+/-0.03 microg protein/ml and another one with a K(d)of 6.57+/-0.87 microg protein/ml. Their B(max)values were 0.54+/-0.11 and 2.34+/-0.39 microg of HDL(3)/mg membrane proteins, respectively. CLA-1, a putative HDL-receptor of 85 kDa, was detected on both BPM and BBM, together with two apo A-l binding sites of 110 and 96 kDa on BPM and BBM, respectively. These results provide further evidence that human placenta possesses specific sites for HDL binding, underlining the important role of maternal HDL in the transfer of cholesterol from the maternal circulation to the placenta and the fetus., (Copyright 1999 Harcourt Publishers Ltd.)

The present investigations should contribute to clarify the importance of beta-adrenergic system in myocard for the triggering of sudden death syndrome and ascites in male broiler chickens. Therefore it should be verified if differences in density of beta-adrenergic receptors in myocard exist in several strains and sexes of chickens. We showed in both male and female broilers that the receptor density was significantly higher as in chickens of the laying strain. There were no significant differences in receptor density between sexes in both investigated strains as well as in KD-values between all groups. The latter finding is referred to the absence of differences in receptor affinity for 3H-dihydroalprenolol between the groups. Clarification of the question if chronic heart failure is in contrast to myocard hypertrophy accompanied with reduction of beta-adrenergic receptor density or receptor affinity in broilers too, as could be shown in other species, has to carried out in further investigations.

Background: Adrenoceptors have been involved in the regulation of hepatocyte proliferation after partial hepatectomy, as well as in primary culture. This report characterizes alpha 1- and beta-adrenoceptor change during the time-course of liver regeneration in adult Wistar Kyoto rats., Methods: Saturation binding assays with [3H]prazosin or [3H]dihydroalprenolol (for alpha 1- and beta-adrenoceptors, respectively) were done in liver plasma membranes from 6-month-old rats subjected to 70% hepatectomy followed by hepatic regeneration., Results: [3H]Prazosin and [3H]dihydroalprenolol binding gave control Bmax values of 101 +/- 10 and 12 +/- 1 fmol/mg protein and Kd of 0.50 +/- 0.10 and 4.1 +/- 0.4 nM for alpha 1- and beta-adrenoceptors, respectively. alpha 1-Adrenoceptor number and Kd increased at 24 and 48 h and returned to control values at 72 and 96 h after surgery, whereas beta-adrenoceptors augmented at 48 and 72 h, with a Kd change at 24 and 48 h posthepatectomy., Conclusions: These results suggest that dual control of alpha 1- and beta-adrenoceptor membrane expression could be involved in different steps during hepatocyte proliferation, and that Wistar Kyoto rats have a different adrenoceptor pattern expression from other rat strains.

The purpose of the present study is to determine the effect of chronic electroconvulsive shock (ECS) on the expression of beta-adrenergic receptors in rat brain by Western blot using mAb beta CO2, a monoclonal antibody against beta-adrenergic receptors. Rats in ECS treated groups received maximal ECS (70 mA, 0.5 second, 60 Hz) through ear-clip electrodes for 12 consecutive days. The experiment was carried out in 14 discrete regions of brain. Chronic ECS reduced the expression of beta-adrenergic receptors in frontal cortex, temporal cortex, parietooccipital cortex, hippocampus and limbic forebrain, but not in other areas of brain. The regional specificity and the magnitude of the reduction of receptor expression are well correlated with those of the reduction of receptor ligand binding, which was determined using [3H]dihydroalprenolol. To the best of our knowledge, this is the first report to demonstrate that chronic ECS decreases the expression of receptor protein in specific regions of rat brain.

DSP-4 is a neurotoxin highly selective for the noradrenergic nerve terminals originating from the locus coeruleus. Preliminary data suggested that its effect in a typical screening test for antidepressant drugs, the forced swimming test, is biphasic dependent on the dose. In the present study, DSP-4 was administered in four doses (5, 10, 30 and 50mg/kg) to male Wistar rats. Administration of the neurotoxin had a dose-dependent biphasic effect on immobility time in the forced swimming test 8 and 9 days later. Thus, DSP-4 at the dose of 10mg/kg increased immobility, but higher doses reduced this measure. The reduction of noradrenaline concentration in the frontal cortex and hippocampus was dose-dependent starting from the dose 10mg/kg. Cortical beta-adrenoceptor binding was increased by DSP-4 treatment at the doses 30mg/kg and 50mg/kg. These results suggest that the increase in immobility time in the forced swimming test is associated with presynaptic changes in noradrenaline availability, whereas the decrease in immobility observed after more complete denervation is associated with postsynaptic receptor supersensitivity.

Changes in the distribution of beta-adrenoceptors (beta ARs) in the plasma membrane and the light vesicle fractions of rat liver during different phases of sepsis were studied using [3H]dihydroalprenolol binding and photoaffinity labeling with [125I]iodocyanopindolol diazirine. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit an initial hypermetabolic (hyperglycemic) phase (9 h after CLP; early sepsis) followed by a hypometabolic (hypoglycemic) phase (18 h after CLP; late sepsis). The radioligand studies show that in the plasma membranes, the density of beta ARs was decreased by 28-32% and 46-69% during the early and the late phases, respectively, of sepsis. In the light vesicles, the density of beta ARs was increased by 25-30% and 30-35% during the early and the late phases, respectively, of sepsis. The total number of the receptor binding sites (the sum of that in plasma membrane plus light vesicle) was decreased by 11-12% and 21-35% during the early and the late phases, respectively, of sepsis. These results indicate that beta ARs were progressively internalized from surface membranes to the intracellular sites and, furthermore, they were underexpressed in the rat liver during the progression of sepsis. Since hepatic glucose metabolism is known to be regulated by catecholamines, in part, through beta AR mediation, an internalization/underexpression of hepatic beta ARs may play a role in the altered glucose homeostasis during sepsis, particularly in the late hypometabolic phase of sepsis.

In most studies, coupling of the beta2-adrenoceptor (beta2AR) to the stimulatory, heterotrimeric GTP-binding protein of adenylyl cyclase the (Gs) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which beta2AR-Gs interactions can be studied directly at the level of the G-protein. We expressed the beta2AR alone, in combination with the alpha-subunit of Gs (G(s alpha)), and as fusion protein with G(s alpha) (beta2AR-G(s alpha)) in Sf9 insect cells. The beta2AR expressed alone couples poorly to the endogenous G(s alpha)-like G-protein of Sf9 cells since no high-affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high-affinity GTPase and guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding were small. Beta2AR-G(s alpha) reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the beta2AR co-expressed with a large excess of G(s alpha). In membranes expressing beta2AR-G(s alpha), highly effective agonist- and inverse agonist regulation of high-affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing beta2AR and G(s alpha) as separate proteins was difficult to detect. Our data show that the beta2AR interacts with G(s alpha) more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused G(s alpha). The beta2AR-G(s alpha) fusion protein provides a very sensitive model system to study the regulation of Gs function by beta2AR agonists and inverse agonists directly at the level of the G-protein.

The role of receptor phosphorylation on the redistribution of beta-adrenergic receptors (beta-ARs) in rat hearts during different phases of sepsis was investigated. Sepsis was induced by cecal ligation and puncture (CLP). Changes in the distribution of beta-ARs in the sarcolemmal and light vesicle fractions were studied using (-)-[4,6-propyl-3H]dihydroalprenolol ([3H]DHA). Phosphorylation of beta-ARs was studied by perfusing hearts with [32P]H3PO4 followed by identification of the phosphorylated beta-ARs with immunoprecipitation using anti-beta 1-AR antibody. The results show that septic rat hearts exhibit an initial hypercardiodynamic (9 h after CLP; early sepsis) and a subsequent hypocardiodynamic (18 h after CLP; late sepsis) state. [3H]DHA binding studies show that, during early sepsis, the maximum binding capacity (Bmax) was increased by 26% in sarcolemma but was decreased by 30% in light vesicles, whereas, during late sepsis, the Bmax was decreased by 39% in sarcolemma but increased by 31% in light vesicles. These data indicate that beta-ARs in the rat heart were externalized from light vesicles to sarcolemma during early sepsis but were internalized from surface membranes to intracellular sites during late sepsis. The immunoprecipitation studies reveal that the externalization of beta-ARs during early sepsis was coupled with a concomitant decrease (-28.5 to -30.6%, P < 0.01) in the receptor phosphorylation, whereas the internalization of beta-ARs during late sepsis was accompanied by a simultaneous increase (30.3 to 33.8%, P < 0.01) in the receptor phosphorylation. Because the phosphorylation/dephosphorylation of beta 1-ARs regulate their functional coupling and may reflect their subcellular distribution, it is suggested that the increase in receptor phosphorylation seen in late sepsis leads to the receptor internalization observed in late sepsis; similarly, externalization of (dephosphorylated) receptors in early sepsis may give rise to the apparent decrease in sarcolemmal receptor phosphorylation observed during this interval.

New Zealand White female rabbits were randomly divided into three groups, each contained six rabbits, i.e. thyroidectomized and untreated rabbits (group 1), thyroidectomized rabbits treated by the Yang tonifying herbs (group 2) and sham thyroidectomized rabbits as controls (group 3). The myocardial beta-adrenoceptor density (Bmax) and affinity (Kd) of each group of rabbits were determined by radioligand binding assay technique on the thirtieth postoperative day and the data were handled by using a computer program of the Woolf plot with weight regression. Moreover, the serum levels of thyroxine (T4) and triiodothyronine (T3) of each group of rabbits were measured by radioimmunoassay technique and their heart rates (HR) were also recorded on the preoperative and thirtieth postoperative day. The results showed that the Bmax, T4, T3 and HR in group 1 were lower significantly than that in group 3 (P < 0.01-0.001), but the change of Kd in group 1 was not significant; the deviation of the indices from the normal value in group 2 was less remarkably than in group 1 other than T4.

The BeWo cell line (b30 clone) has been examined as a potential in vitro system to study transplacental transport. At the light and electron microscope level, the cells were observed to form confluent monolayers on polycarbonate filters in approximately 5 days and morphologically resembled the typical human trophoblast. BeWo monolayers developed a modest transepithelial electrical resistance and a molecular size-dependent permeability to hydrophilic passive diffusion markers, fluorescein, and selected fluorescein-labeled dextrans. Linoleic acid permeation across BeWo monolayers was asymmetric, saturable, and inhibited by low temperature and excess competing fatty acid. Forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate treatments stimulated morphological changes in BeWo cultures and enhanced the asymmetric passage of linoleic acid across the BeWo monolayers while having minimal effects on passive permeability, affirming that the differentiation state of the cells can influence membrane transporters and transmonolayer permeability. The basic permeability properties of the BeWo monolayers suggest that the cells grown on permeable supports may be examined as a convenient in vitro system to evaluate some transplacental transport mechanisms.

Unlabelled: Local anesthetics inhibit binding of ligands to beta2-adrenergic receptors (beta2ARs), and, as a consequence, inhibit intracellular cAMP production. We hypothesized that among homologous local anesthetics, their avidity at inhibiting binding of tritiated dihydroalprenolol (3H-DHA) to beta2ARs would increase with increasing length of alkyl substituents and would demonstrate stereospecificity. Specific binding of 3H-DHA to human beta2ARs was assayed in the presence of six different members of the 1-alkyl-2,6-pipecoloxylidide class of local anesthetics (including mepivacaine, ropivacaine, and bupivacaine), the R(+) and S(-) bupivacaine enantiomers, lidocaine, prilocaine, etidocaine, procaine, and tetracaine. Avidity of binding to beta2ARs increased with increasing length of the alkyl chain (pKi values = 2.4, 3.6, 4.3, 4.1, 4.1, 5.9 for the methyl [mepivacaine], ethyl, S(-)propyl [ropivacaine], butyl [bupivacaine], pentyl, and octyl derivatives, respectively). We found no evidence for bupivacaine stereospecificity (pKi values = 4.3 and 4.9 for the S(-) and R(+) isomers, respectively). Other amide and ester local anesthetics also showed increasing potency with increasing length of alkyl substituents (pKi values = 3.6, 3.8, and 4.3 for lidocaine, prilocaine, and etidocaine; 4.2 and 5.6 for procaine and tetracaine, respectively). The correlation between increased inhibition of beta2AR binding and alkyl chain length resembles the correlation between local anesthetic potency at nerve block and increased alkyl chain length. The lack of clear stereospecificity is consistent with the relatively low potency these agents demonstrate at inhibition of beta2AR binding. Finally, the relatively potent inhibition of beta2ARs by etidocaine, tetracaine, and bupivacaine suggests that their propensity for cardiovascular depression after accidental intravenous overdose could result from beta2AR or beta1AR blockade and inhibition of cAMP production., Implications: Local anesthetics demonstrate a rank order of avidity for displacing ligands from beta2-adrenergic receptors such that larger molecules displace ligands at lower concentrations than smaller local anesthetic molecules. This relationship between molecular size and receptor avidity could explain the greater propensity for cardiovascular toxicity of relatively large local anesthetics such as bupivacaine.

To study whether the changes in cortical noradrenergic and serotonergic mechanisms observed in patients with Alzheimer's disease are the consequence of reduced cortical cholinergic activity, a novel colinergic immunotoxin (conjugate of the monoclonal antibody 192IgG against the lower affinity nerve growth factor receptor with the cytotoxic protein saporin, 192IgG-saporin) was used to produce a specific and selective loss of cholinergic cells in rat basal forebrain nuclei. To correlate the responses to cholinergic immunolesion in cholinoceptive cortical target regions with cholinergic hypoactivity, quantitative receptor autoradiography to measure adrenoceptors and 5-hydroxytryptamine (5-HT) receptor subtypes, and histochemistry to estimate acetylcholinesterase activity, were performed in adjacent brain sections. alpha 1-adrenoceptor and 5-HT1A receptor binding were not affected by cholinergic immunolesion in any of the cortical and hippocampal regions studied. However, cholinergic immunolesion resulted in significantly reduced alpha 2- and beta-adrenoceptor as well as 5-HT2A receptor binding in a number of cortical and hippocampal regions displaying a reduced activity of acetylcholinesterase, already detectable seven days after a single injection of 192IgG-saporin and persisting up to three months post lesion without any significant recovery. The data suggest that at least a subpopulation of alpha 2- and beta-adrenoceptor as well 5-HT2A receptor subtype is present on cortical and hippocampal cholinergic terminals originating in the basal forebrain. The lesion-induced receptor changes suggest that the alterations in cortical 5-HT2 receptor binding observed in patients with Alzheimer's disease might be secondary to cholinergic deficits.

Renal nerve stimulation or circulating catecholamines activate the beta-adrenergic receptors that mediate direct effects on tubular transport. Three subtypes of beta-adrenergic receptors have been characterized: beta 1, beta 2, and beta 3. beta-Adrenergic-receptor effects on Na+ and Ca2+ transport in distal convoluted tubules (DCT) have not been established. The focus of this study was to 1) identify the subtypes of beta-adrenergic receptors in DCT cells and 2) examine functional responses to beta-receptor activation on adenosine 3',5'-cyclic monophosphate (cAMP) formation and Na+ and Ca2+ entry. To determine the subtypes of beta-receptors present, RNA isolated from immortalized mouse DCT cells was reverse transcribed, and the cDNA was amplified using primers designed to reported sequences for beta 1-, beta 2-, and beta 3-receptor subtypes. Products of the appropriate sizes were obtained with beta 1- and beta 2-primers. No product was observed with primers to the beta 3 sequence. Receptor products were confirmed by sequencing and are identical to reported mouse beta 1- and beta 2-receptor sequence. Receptor binding of[3H]dihydroalprenolol was 123 +/- 13 fmol/mg protein, and a 3:1 ratio of beta 1- to beta 2-receptors was observed with DCT cell membranes. Isoproterenol, a beta-receptor agonist, increased cAMP formation 8.5-fold. Pretreatment with the antagonist propranolol abolished agonist-induced cAMP accumulation. Isoproterenol significantly increased 22Na+ uptake to 345 +/- 23 compared with a basal rate of 256 +/- 12 nmol.min-1.mg protein-1 and was blocked with propranolol and beta 1- and beta 2-selective antagonists. Isoproterenol had no effect on 45Ca2+ entry into DCT cells. In summary, DCT cells express three times more beta 1- than beta 2-receptors and express no detectable beta 3-adrenergic receptors. beta-Receptors couple to adenylyl cyclase, and activation of beta-adrenergic receptors increases Na+ but not Ca2+ entry in DCT cells.

Thyroid hormone (triiodothyronine; T3) has been shown to control the expression of beta 1-adrenergic receptors (beta 1-AR) in cardiac myocytes, but not in C6 glioma cells. This cell specificity has been attributed to low expression of T3 receptors and high expression of the c-erbA alpha 2 splice variant that interferes with the action of T3. To check this hypothesis we have expressed the c-erbA/thyroid hormone receptor (TR) alpha 1 gene in C6 glioma cells and investigated their response to thyroid hormone. Cells expressing TR alpha 1, but not wild-type cells, were responsive to T3 as shown by increased expression of mitochrondrial hydroxymethylglutaryl CoA synthase after T3 exposure. However, T3 had no effect on beta 1-AR gene expression in either set of cells. The beta 1-AR mRNA concentrations were, however, altered by retinoic acid (RA) treatment. Retinoic acid caused a rapid up-regulation of beta 1-AR mRNA levels that was blocked by cycloheximide. Retinoic acid did not increase the beta 1-AR gene transcription rate in run-on experiments. These results indicate an indirect post-transcriptional effect of RA. Control of beta 1-AR expression in C6 cells is also exerted at the translational level, because there was no correlation between mRNA and protein induction, as determined by radioligand binding studies. We conclude that lack of responsiveness of the beta 1-AR gene in C6 cells to T3 is not due to high expression of c-erbA alpha 2 but to undefined cell-specific factors.

The beta-adrenoceptors present in dog liver were characterized by radioligand binding techniques. [3H]Dihydroalprenolol bound with high affinity (KD 0.9 nM) to a relatively low number of sites (approximately 68 fmol/mg protein) in partially purified dog liver plasma membranes. These sites were characterized by binding competition; the orders of affinity were: (a) for agonists: (-)isoproterenol > or = salbutamol > epinephrine > or = terbutaline > norepinephrine > (+)isoproterenol and (b) for antagonists: propranolol > ICI 118551 >> metoprolol >> atenolol. These data indicate that dog liver expresses beta-adrenoceptors of the beta2 subtype.

The ultra-short-acting and vasorelaxant beta 1-adrenoceptor blocking activities of vasomolol, a guaiacoxypropanolamine derivative of vanillic acid ethyl ester, were studied. Vasomolol (0.5, 1.0, 3.0 mg/kg intravenously, i.v.) produced a dose-dependent bradycardia response and demonstrated particularly a hypotensive action with an ultra-short-acting property in pentobarbital-anesthetized normotensive rats. Vasomolol's steady state of beta-blockade was attained < or = 10 min after initial infusion, and a rapid recovery from blockade occurred after discontinuation of the infusion, although intravenous infusion of vasomolol (300 micrograms/kg/min) could not inhibit pressor responses induced by (-)phenylephrine (10 micrograms/kg i.v.). In isolated rat thoracic aorta, vasomolol (1-10 microM) inhibited vascular smooth muscle contractions induced by both (-)phenylephrine (10(-5) M) and high K+ (75 mM) concentration dependently. This inhibitory effect of vasomolol was more sensitive on K(+)-induced than on (-)phenylephrine-induced contractions, suggesting that the block of Ca2+ influx may involve the major mechanism of vasorelaxation. In isolated guinea pig tissues, vasomolol (0.01-10 microM) antagonized the (-)isoproterenol (ISO)-induced positive inotropic and chronotropic effects of the atria and tracheal relaxation responses in a concentration-dependent manner. The parallel shift to the right of the concentration-response curve of (-)ISO suggested that vasomolol was a beta-adrenoceptor competitive antagonist. The effect of vasomolol was more potent on atria than on tracheal tissues, indicating that it possesses beta 1-adrenoceptor selectivity. In addition, vasomolol did not show intrinsic sympathomimetic activity (ISA). Moreover, the binding characteristics of vasomolol were evaluated in [3H]dihydroalprenolol ([3H]DHA) binding to porcine ventricular membranes. Vasomolol was an ultra-short-acting and highly selective beta 1-adrenoceptor antagonist with vasorelaxant activity and is devoid of ISA.