Cardiovascular and metabolic alterations in mice lacking both beta1- and beta2-adrenergic receptors.

The activation state of beta-adrenergic receptors (beta-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by beta-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of beta1- and/or beta2-AR expression on these homeostatic control mechanisms. Despite total absence of beta1- and beta2-ARs, the predominant cardiovascular beta-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of beta-AR function by beta-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in beta1/beta2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single beta1- and beta2-AR knockouts as well as the beta1-/beta2-AR double knock-out suggest that in the mouse, beta-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the beta1-AR, whereas vascular relaxation and metabolic rate are controlled by all three beta-ARs (beta1-, beta2-, and beta3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular beta3-AR responsiveness are also observed in beta1-/beta2-AR double knockouts. In addition to its ability to define beta-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed.     

Expression and functional analysis of beta-adrenoceptor subtypes in rabbit submandibular gland

beta-Adrenoceptors (beta-ARs) mediate important physiological functions in salivary glands. Here we investigated the expression and function of beta-AR subtypes in rabbit submandibular gland (SMG). Both beta(1)- and beta(2)-ARs, but not beta(3)-AR, were strongly expressed in rabbit SMG. beta(1)-AR proteins were widely expressed in acinar and ductal cells whereas beta(2)-AR proteins were mainly detected in ductal cells. A [(3)H]-dihydroalprenolol binding assay revealed that beta-AR B(max) was 186+/-11.9 fmol/mg protein and K(d) was 2.71+/-0.23 nM. A competitive binding assay with CGP 20712A, a beta(1)-AR antagonist, indicated that the proportion of beta(1)-AR and beta(2)-AR was 71.9% and 28.1%, respectively. Gland perfusion with the beta-AR agonist isoproterenol induced a significant increase in saliva secretion which was abolished by pretreatment with the non-selective beta-AR antagonist propranolol. Pretreatment with beta(1)- or beta(2)-AR selective antagonists, CGP 20712A or ICI 118551, diminished isoproterenol-induced increase in saliva secretion by 71.2% and 28.8%, respectively. The expression of alpha-amylase mRNA was significantly stimulated by isoproterenol, which was eliminated by propranolol and CGP 20712A. Perfusion with isoproterenol decreased alpha-amylase protein storage in SMG and increased alpha-amylase activity in saliva. These alterations became less significant after pretreatment with propranolol and CGP 20712A. Our results suggest that both beta(1)- and beta(2)-ARs are expressed in rabbit SMG. beta(1)-AR is the predominant subtype and may play an important role in regulating saliva and alpha-amylase secretion.     

Colorectal cancer metastases affect the biochemical characteristics of the human liver beta-adrenoceptor-G-protein-adenylate cyclase system

The sympathetic-catecholamine system is involved in the regulation of hepatic metabolic pathways mainly through cAMP-linked beta2-adrenoceptors (beta2-ARs) in humans and to a lesser extent through cAMP-independent mechanisms, but no information is available about the possible biochemical changes of beta2-ARs and their signalling pathways in human colorectal cancer (CRC) and colorectal cancer hepatic metastases (CRCHM). Changes in density and distribution of beta-ARs as well as in post-receptor signalling components were studied in membranes of human liver with CRCHM, and for comparison, in membranes of nonadjacent, non-metastatic human liver (NA-NM) obtained from 13 patients, using binding and competition binding studies. Studies were also carried out using normal and cancerous human colon tissues. In CRCHM, the density of beta-ARs (B(max)) was significantly reduced, compared to NA-NM liver tissues (40.09+/-2.83 vs. 23.09+/-3.24 fmol/mg protein; P<0.001). A similar decrease in the beta-AR density was observed in the colon with primary colorectal cancer compared to healthy colon (37.6+/-2.2 vs. 23.8+/-3.5 fmol/mg protein), whereas the affinity of ICYP binding to the receptor remained unaffected. Desensitized beta-ARs were uncoupled from stimulatory G-protein (G(S)), as total density of beta-adrenoceptors in the high affinity state was significantly reduced. Concomitantly, CRCHM elicited decrease in the catalytic adenylate cyclase (AC) activity (cAMP formation) in response to isoproterenol plus GTP or forskolin or NaF. In NA-NM and CRCHM liver, the inhibition-concentration curves of ICI 118.551 showed the presence of a homogeneous population of the beta2-AR subtypes. Neither the binding patterns nor the inhibition constant (K(i)) of ICI 118.551 were altered in CRCHM. In CRCHM, the hepatic beta-AR-G-protein(s)-AC signalling system was markedly impaired, thus, these changes may well influence beta-AR-mediated functions in both organs.     

Activity of the unique beta-adrenergic Na+/H+ exchanger in trout erythrocytes is controlled by a novel beta3-AR subtype

beta-Adrenoceptors (beta-ARs) are seven-transmembrane domain, G protein-coupled receptors that transduce the cellular effects of epinephrine and norepinephrine and play a pivotal role in the vertebrate stress response. This study reports the cloning and characterization of two previously unreported beta-ARs from the rainbow trout (Oncorhynchus mykiss). Phylogenetic analysis of amino acid sequences indicates that both beta-ARs are homologs of the mammalian beta3-AR. Analysis of tissue expression patterns indicates that one of these trout beta3-adrenoceptors (beta3a-AR) is highly expressed in gill and heart, whereas the second (beta3b-AR) is highly expressed by red blood cells (RBC). Expression of the beta3b-AR in the RBC coupled with the finding of a single category of beta-AR binding sites on RBC membranes provides strong evidence for the control of the trout RBC beta-AR Na+/H+ exchanger (beta-NHE) activity by signaling through this beta3b-subtype and not through a beta1-subtype as previously proposed. The RBC-specific trout beta3b-AR exhibits binding characteristics that distinguish this receptor from each of the three pharmacologically defined categories of mammalian beta-ARs (beta1-, beta2-, and beta3-AR). This study is the first to report the presence of a beta3-AR subtype in a fish species, and the proposal that the beta3b-AR controls RBC beta-NHE activity represents a novel role for the beta3-AR subtype in vertebrates.     

Expression of human alpha 2-adrenergic receptors in adipose tissue of beta 3-adrenergic receptor-deficient mice promotes diet-induced obesity

Catecholamines play an important role in controlling white adipose tissue function and development. beta- and alpha 2-adrenergic receptors (ARs) couple positively and negatively, respectively, to adenylyl cyclase and are co-expressed in human adipocytes. Previous studies have demonstrated increased adipocyte alpha 2/beta-AR balance in obesity, and it has been proposed that increased alpha 2-ARs in adipose tissue with or without decreased beta-ARs may contribute mechanistically to the development of increased fat mass. To critically test this hypothesis, adipocyte alpha 2/beta-AR balance was genetically manipulated in mice. Human alpha 2A-ARs were transgenically expressed in the adipose tissue of mice that were either homozygous (-/-) or heterozygous (+/-) for a disrupted beta 3-AR allele. Mice expressing alpha 2-ARs in fat, in the absence of beta 3-ARs (beta 3-AR -/- background), developed high fat diet-induced obesity. Strikingly, this effect was due entirely to adipocyte hyperplasia and required the presence of alpha2-ARs, the absence of beta 3-ARs, and a high fat diet. Of note, obese alpha 2-transgenic beta 3 -/- mice failed to develop insulin resistance, which may reflect the fact that expanded fat mass was due to adipocyte hyperplasia and not adipocyte hypertrophy. In summary, we have demonstrated that increased alpha 2/beta-AR balance in adipocytes promotes obesity by stimulating adipocyte hyperplasia. This study also demonstrates one way in which two genes (alpha 2 and beta 3-AR) and diet interact to influence fat mass.     

Overexpression of beta2-adrenergic receptors in mouse liver alters the expression of gluconeogenic and glycolytic enzymes

In the livers of humans and many other mammalian species, beta2-adrenergic receptors (beta2-ARs) play an important role in the modulation of glucose production by glycogenolysis and gluconeogenesis. In male mice and rats, however, the expression and physiological role of hepatic beta2-ARs are rapidly lost with development under normal physiological conditions. We previously described a line of transgenic mice, F28 (Andre C, Erraji L, Gaston J, Grimber G, Briand P, and Guillet JG. Eur J Biochem 241: 417-424, 1996), which carry the human beta2-AR gene under the control of its own promoter. In these mice, hepatic beta2-AR levels are shown to increase rapidly after birth and, as in humans, be maintained at an elevated level in adulthood. F28 mice display strongly enhanced adenylyl cyclase responses to beta-AR agonists in their livers and, compared with normal mice, have increased basal hepatic adenylyl cyclase activity. In this report we demonstrate that, under normal physiological conditions, this increased beta2-AR activity affects the expression of the gluconeogenic and glycolytic key enzymes phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and l-pyruvate kinase and considerably decreases hepatic glycogen levels. Furthermore, we show that the effects of beta-adrenergic ligands on liver glycogen observed in humans are reproduced in these mice: liver glycogen levels are strongly decreased by the beta2-AR agonist clenbuterol and increased by the beta-AR antagonist propranolol. These transgenic mice open new perspectives for studying in vivo the hepatic beta2-AR system physiopathology and for testing the effects of beta-AR ligands on liver metabolism.     

Coupling of beta-adrenergic receptors to cardiac L-type Ca2+ channels: preferential coupling of the beta1 versus beta2 receptor subtype and evidence for PKA-independent activation of the channel.

Beta1- and beta2-adrenergic receptors (beta-ARs) co-exist in mammalian heart, and it is generally accepted that both activate adenylyl cyclase (AC), resulting in increased levels of cAMP and subsequent activation of L-type Ca2+ channels (CaCh). To investigate the contribution of each beta-AR subtype in AC and CaCh coupling, we stably expressed cardiac CaCh alpha1 and beta2 subunits along with either beta1-AR or beta2-AR in CHW fibroblasts. Co-expression of either beta-AR with CaCh subunits conferred responsiveness of AC and CaCh to isoproterenol (ISO), which was not observed in non-transfected cells. ISO-promoted cAMP formation occurred at a lower EC50 through the beta2-AR than through the beta1-AR (0.13 +/- 0.01 vs. 0.6 +/- 0.14 nM). In contrast, activation of CaCh was more efficacious via the beta1-AR than the beta2-AR (EC50 for CaCh activation = 238 +/- 33 vs. 1057 +/- 113 nM). Pre-treatment with pertussis toxin (PTX) had no effect upon the responsiveness of either cAMP formation or CaCh activation through either receptor. We conclude (1) that beta1-ARs exhibit preferential coupling to CaCh activation, versus that observed for the beta2-AR; (2) that this preferential coupling cannot be explained solely by cAMP-dependent processes; and (3) that the relative attenuation of beta2-AR-promoted CaCh activation is not due to receptor coupling to PTX-sensitive G proteins. Thus, it is likely that other subtype-specific, cAMP-independent coupling of the beta-AR to CaCh is present.     

Regulation of the rainbow trout (Oncorhynchus mykiss) hepatic beta2-adrenoceptor by adrenergic agonists

The characteristics of hepatic beta(2)-adrenoceptors (AR) were examined in rainbow trout (Oncorhynchus mykiss) chased once per day to exhaustion for up to 7 days or fed the repartitioning agents clenbuterol (CLEN) or ractopamine (RACT) that function in mammals as beta-agonists. A one-day chase and feeding the CLEN for 37 days resulted in a significant 27% and 33% decrease, respectively, in the number of CGP-binding sites (B(max)) with no significant change in affinity (Kd) of hepatic beta(2)-ARs. Despite the significant decrease in beta(2)-AR numbers with CLEN feeding, no significant differences were found for either beta(2)-AR mRNA levels or adenylyl cyclase (ACase) activities. In addition, CLEN displayed only partial agonist activities as it was found to be more effective at blocking isoproterenol-stimulated cAMP production in isolated hepatocytes than stimulating cAMP production. The small affects of RACT may be related to its low active stereoisomer content and low affinity for the trout beta(2)-AR. Agonist regulation of the trout hepatic beta(2)-ARs may involve down-regulation of the receptors without affecting responsiveness.     

Beta 2-adrenergic receptor signaling acts via NO release to mediate ACh-induced activation of ATP-sensitive K+ current in cat atrial myocytes.

In atrial myocytes, an initial exposure to isoproterenol (ISO) acts via cAMP to mediate a subsequent acetylcholine (ACh)-induced activation of ATP-sensitive K(+) current (I(K,ATP)). In addition, beta-adrenergic receptor (beta-AR) stimulation activates nitric oxide (NO) release. The present study determined whether the conditioning effect of beta-AR stimulation acts via beta(1)- and/or beta(2)-ARs and whether it is mediated via NO signaling. 0.1 microM ISO plus ICI 118,551 (ISO-beta(1)-AR stimulation) or ISO plus atenolol (ISO-beta(2)-AR stimulation) both increased L-type Ca(2+) current (I(Ca,L)) markedly, but only ISO-beta(2)-AR stimulation mediated ACh-induced activation of I(K,ATP). 1 microM zinterol (beta(2)-AR agonist) also increased I(Ca,L) and mediated ACh-activated I(K,ATP). Inhibition of NO synthase (10 microM L-NIO), guanylate cyclase (10 microM ODQ), or cAMP-PKA (50 microM Rp-cAMPs) attenuated zinterol-induced stimulation of I(Ca,L) and abolished ACh-activated I(K,ATP). Spermine-NO (100 microM; an NO donor) mimicked beta(2)-AR stimulation, and its effects were abolished by Rp-cAMPs. Intracellular dialysis of 20 microM protein kinase inhibitory peptide (PKI) abolished zinterol-induced stimulation of I(Ca,L). Measurements of intracellular NO ([NO](i)) using the fluorescent indicator DAF-2 showed that ISO-beta(2)-AR stimulation or zinterol increased [NO](i). L-NIO (10 microM) blocked ISO- and zinterol-induced increases in [NO](i). ISO-beta(1)-AR stimulation failed to increase [NO](i). Inhibition of G(i)-protein by pertussis toxin significantly inhibited zinterol-mediated increases in [NO](i). Wortmannin (0.2 microM) or LY294002 (10 microM), inhibitors of phosphatidylinositol 3'-kinase (PI-3K), abolished the effects of zinterol to both mediate ACh-activated I(K,ATP) and stimulate [NO](i). We conclude that both beta(1)- and beta(2)-ARs stimulate cAMP. beta(2)-ARs act via two signaling pathways to stimulate cAMP, one of which is mediated via G(i)-protein and PI-3K coupled to NO-cGMP signaling. Only beta(2)-ARs acting exclusively via NO signaling mediate ACh-induced activation of I(K,ATP). NO signaling also contributes to beta(2)-AR stimulation of I(Ca,L). The differential effects of beta(1)- and beta(2)-ARs can be explained by the coupling of these two beta-ARs to different effector signaling pathways.     

Molecular modeling of SWR-0342SA, a beta3-selective agonist, with beta1- and beta3-adrenoceptor

The molecular dynamics (MD) simulations study in the formation of the complex between compound SWR-0342SA and beta-ARs suggested that upon binding SWR-0342SA stimulates receptor activation through residues network (Asp104, Leu335 in beta(1)-AR; Asp117, Ser209, Leu303, Ser191 in beta(3)-AR) in an active conformation state. The models suggest that the structural origin of the selectivity of SWR-0342SA to beta(3)-AR vs. beta(1)-AR comes from the following results: (a) the tight interaction between the agonist and the TMs 3, 5, 6 and 2 nd EC loop. Asp117 interacts with the cationic amino group of the agonist molecule. (b) Additional contacts are done with Ser209, Leu303 and Ser191. These results are in good agreement with the binding affinities (pKi values) of SWR-0342SA to beta-AR family expressed in recombinant mammalian cells.     

Overexpression of beta2-adrenergic receptors cAMP-dependent protein kinase phosphorylates and modulates slow delayed rectifier potassium channels expressed in murine heart: evidence for receptor/channel co-localization

The cardiac slow delayed rectifier potassium channel (IKs), comprised of (KCNQ1) and beta (KCNE1) subunits, is regulated by sympathetic nervous stimulation, with activation of beta-adrenergic receptors PKA phosphorylating IKs channels. We examined the effects of 2-adrenergic receptors (beta2-AR) on IKs in cardiac ventricular myocytes from transgenic mice expressing fusion proteins of IKs subunits and hbeta2-ARs. KCNQ1 and beta2-ARs were localized to the same subcellular regions, sharing intimate localization within nanometers of each other. In IKs/B2-AR myocytes, IKs density was increased, and activation shifted in the hyperpolarizing direction; IKs was not further modulated by exposure to isoproterenol, and KCNQ1 was found to be PKA-phosphorylated. Conversely, beta2-AR overexpression did not affect L-type calcium channel current (ICaL) under basal conditions with ICaL remaining responsive to cAMP. These data indicate intimate association of KCNQ1 and beta2-ARs and that beta2-AR signaling can modulate the function of IKs channels under conditions of increased beta2-AR expression, even in the absence of exogenous beta-AR agonist.     

Modification of beta-adrenoceptor signal transduction pathway by genetic manipulation and heart failure

The beta-adrenoceptor (beta-AR) mediated signal transduction pathway in cardiomyocytes is known to involve beta1- and beta2-ARs, stimulatory (Gs) and inhibitory (Gi) guanine nucleotide binding proteins, adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). The activation of beta1- and beta2-ARs has been shown to increase heart function by increasing Ca2+ -movements across the sarcolemmal membrane and sarcoplasmic reticulum through the stimulation of Gs-proteins, activation of AC and PKA enzymes and phosphorylation of the target sites. The activation of PKA has also been reported to increase phosphorylation of some myofibrillar proteins (for promoting cardiac relaxation) and nuclear proteins (for cardiac hypertrophy). The activation of beta2-AR has also been shown to affect Gi-proteins, stimulate mitogen activated protein kinase and increase protein synthesis by enhancing gene expression. Beta1- and beta2-ARs as well as AC are considered to be regulated by PKA- and protein kinase C (PKC)-mediated phosphorylations directly; both PKA and PKC also regulate beta-AR indirectly through the involvement of beta-AR kinase (betaARK), beta-arrestins and Gbeta gamma-protein subunits. Genetic manipulation of different components and regulators of beta-AR signal transduction pathway by employing transgenic and knockout mouse models has provided insight into their functional and regulatory characteristics in cardiomyocytes. The genetic studies have also helped in understanding the pathophysiological role of PARK in heart dysfunction and therapeutic role of betaARK inhibitors in the treatment of heart failure. Varying degrees of defects in the beta-AR signal transduction system have been identified in different types of heart failure to explain the attenuated response of the failing heart to sympathetic stimulation or catecholamine infusion. A decrease in beta1-AR density, an increase in the level of G1-proteins and overexpression of betaARK are usually associated with heart failure; however, these attenuations have been shown to be dependent upon the type and stage of heart failure as well as region of the heart. Both local and circulating renin-angiotensin systems, sympathetic nervous system and endothelial cell function appears to regulate the status of beta-AR signal transduction pathway in the failing heart. Thus different components and regulators of the beta-AR signal transduction pathway appears to represent important targets for the development of therapeutic interventions for the treatment of heart failure.     

Targeted disruption of the beta2 adrenergic receptor gene.

beta-Adrenergic receptors (beta-ARs) are members of the superfamily of G-protein-coupled receptors that mediate the effects of catecholamines in the sympathetic nervous system. Three distinct beta-AR subtypes have been identified (beta1-AR, beta2-AR, and beta3-AR). In order to define further the role of the different beta-AR subtypes, we have used gene targeting to inactivate selectively the beta2-AR gene in mice. Based on intercrosses of heterozygous knockout (beta2-AR +/-) mice, there is no prenatal lethality associated with this mutation. Adult knockout mice (beta2-AR -/-) appear grossly normal and are fertile. Their resting heart rate and blood pressure are normal, and they have a normal chronotropic response to the beta-AR agonist isoproterenol. The hypotensive response to isoproterenol, however, is significantly blunted compared with wild type mice. Despite this defect in vasodilation, beta2-AR -/- mice can still exercise normally and actually have a greater total exercise capacity than wild type mice. At comparable workloads, beta2-AR -/- mice had a lower respiratory exchange ratio than wild type mice suggesting a difference in energy metabolism. beta2-AR -/- mice become hypertensive during exercise and exhibit a greater hypertensive response to epinephrine compared with wild type mice. In summary, the primary physiologic consequences of the beta2-AR gene disruption are observed only during the stress of exercise and are the result of alterations in both vascular tone and energy metabolism.     

Up-regulation of uncoupling proteins by beta-adrenergic stimulation in L6 myotubes

Catecholamine-induced and beta-adrenergic receptor (beta-AR)-mediated thermogenesis in skeletal muscle is a significant component of whole-body energy expenditure. Skeletal muscle expresses uncoupling protein (UCP) 2 and UCP3, which can dissipate the transmitochondrial electrochemical gradient and thereby may be involved in regulation of energy metabolism. We investigated the effects of beta-AR stimulation on UCP2 and UCP3 expression in L6 myotubes. Stimulation of the cells with epinephrine increased the UCP3 mRNA level transiently at 6 h, and also the UCP2 mRNA level at 6-24 h. The stimulatory effects of epinephrine were also observed in the presence of carbacyclin and 9-cis retinoic acid, and mimicked by isoproterenol and salbutamol (beta2-AR agonists), but abolished by propranolol and ICI-118,551 (beta2-AR antagonists). Pharmacological and mRNA analyses revealed the existence of beta2-AR, but not beta1- and beta3-ARs, in L6 myotubes. These results suggested that catecholamines up-regulate UCP2 and UCP3 expression through direct action on the beta2-AR in skeletal muscle.     

G(i)-dependent suppression of beta(1)-adrenoceptor effects in ventricular myocytes from NE-treated guinea pigs

It has been suggested that there is a preferential coupling in heart muscle between the inhibitory G protein (G(i)) and the beta(2)-subtype of the beta-adrenergic receptor (beta-AR), since pertussis toxin (which inactivates G(i)) reveals latent beta(2)-ARs in rat and mouse myocytes. We have previously shown that guinea pigs treated with norepinephrine (NE) for 7 days have myocytes that are desensitized to beta-AR-agonist stimulation, and that pertussis toxin restores these responses. The purpose of the present investigation was to determine whether pertussis toxin specifically upregulated beta(2)-ARs in myocytes from NE-treated guinea pigs. The sole beta-AR subtype in control guinea pig myocytes was confirmed as beta(1)-AR by radioligand binding, single-cell autoradiography, and concentration-response curves to isoproterenol in contracting myocytes. In contrast, a minor pool of beta(2)-ARs was observed in rat myocytes by use of the same methods. NE treatment decreased the maximum isoproterenol response (relative to high Ca(2+)) from 0.89 +/- 0.06 to 0.58 +/- 0.08 (n = 7, P < 0.01) and the pD(2) (-log EC(50)) from 8.8 +/- 0.2 to 7.5 +/- 0.2 (n = 7, P < 0.01). Pertussis toxin treatment increased the isoproterenol-to-Ca(2+) ratio to 0.88 +/- 0.04 (n = 6, P < 0.05) and the pD(2) to 8.6 +/- 0.3 (P < 0.01). This was not mediated by increases in either number or function of beta(2)-ARs. G(i) is therefore able to modulate beta(1)-AR responses in guinea pig myocytes.     

Synthesis and first in vivo evaluation of new selective high affinity beta1-adrenoceptor radioligands for SPECT based on ICI 89,406

The results of cardiac biopsies suggest that myocardial beta1-adrenoceptor (AR) density is reduced in patients with chronic heart failure, while changes in cardiac beta2-ARs vary. A technique for visualization and quantification of beta1-AR populations rather than total beta-AR densities in the human heart would be of great clinical interest. Molecular imaging techniques, either single photon emission computed tomography (SPECT) or positron emission tomography (PET), with appropriate radiopharmaceuticals offer the possibility to assess beta-AR density noninvasively in humans, but to date, neither a SPECT nor a PET-radioligand is clinically established for the selective imaging of cardiac beta1-ARs. The aim of this study was to design a high affinity selective beta1-AR radioligand for the noninvasive in vivo imaging of cardiac beta1-AR density in man using SPECT. Based on the well-known selective beta1-AR antagonist, ICI 89,406, both the racemic iodinated target compound 11a and the (S)-enantiomer 15a were synthesized. Competition studies using the nonselective AR ligand, [(125)I]iodocyanopindolol ([(125)I]ICYP), and ventricular membrane preparations from mice showed that 11a and 15a possess higher beta1-AR affinities (up to 265-fold) and beta1-AR selectivities (up to 245-fold) than ICI 89,406. Encouraged by these results, the radioiodinated counterparts of racemic 11a (11b: (125)I, 11c: (123)I) and (S)-configurated 15a (15b: (125)I, 15c: (123)I) were synthesized. The target compounds were evaluated in rats. Biodistribution and metabolism studies in rats indicated that there is a specific heart uptake of 11b-c and especially 15b-c accompanied by rapid metabolism of the radioligands. Therefore, radioiodinated 11c and 15c appeared to be unpromising SPECT-radioligands for assessing beta1-ARs in vivo in the rat. However, the rat may metabolize beta-AR ligands more rapidly than other species as demonstrated for (S)-[(11)C]CGP 12177, a radioligand structurally related to 11a-c and 15a-c. Therefore further studies in a different animal model will be carried out.     

Are developing beta-adrenoceptors able to desensitize? Acute and chronic effects of beta-agonists in neonatal heart and liver

During fetal and neonatal development, beta-adrenergic receptors (beta-ARs) appear to be resistant to desensitization by beta-agonist drugs. To determine the mechanisms underlying the regulatory differences between adults and neonates, we administered isoproterenol, a mixed beta(1)/beta(2)-AR agonist, and terbutaline, a beta(2)-selective agonist. Effects were examined in the ensuing 4 h after a single injection, or after the last of four daily injections. We prepared cell membranes from heart (predominantly beta(1)-ARs) and liver (predominantly beta(2)-ARs) and assessed signal transduction in the adenylyl cyclase (AC) pathway. In the first few hours after a single administration of isoproterenol to adult rats, cardiac beta-ARs showed activation of G proteins (elevated AC response to forskolin) and desensitization of beta-AR-mediated responses; after the fourth injection, heterologous desensitization emerged, characterized by a loss of signaling mediated either through beta-ARs or glucagon receptors. Terbutaline evoked an increase in the forskolin response but no desensitization of receptor-mediated responses. When we gave the same treatments to neonatal rats, we observed cardiac G protein activation, but there was neither homologous nor heterologous desensitization of beta-ARs or glucagon receptors. In the adult liver, isoproterenol and terbutaline both failed to evoke desensitization, regardless of whether the drugs were given once or for 4 days. In neonates, however, acute or chronic treatment elicited homologous desensitization of beta-AR-mediated AC signaling, while sensitizing the response to glucagon. These results show that neonatal beta-ARs are inherently capable of desensitization in some, but not all, cell types; cellular responses can be maintained through heterologous sensitization of signaling proteins downstream from the receptor. Differences from adult patterns of response are highly tissue selective and are likely to depend on ontogenetic differences in subtypes of beta-ARs and AC.     

Atypical beta-adrenergic receptor in 3T3-F442A adipocytes. Pharmacological and molecular relationship with the human beta 3-adrenergic receptor.

Expression of ligand binding properties for an atypical beta-adrenergic receptor (beta-AR) subtype was studied during the adipose differentiation of murine 3T3-F442A cells and compared with that of the human beta 3-AR expressed in Chinese hamster ovary cells stably transfected with the human beta 3-AR gene (CHO-beta 3 cells) Emorine, L. J., Marullo, S., Briend-Sutren, M. M., Patey, G., Tate, K., Delavier-Klutchko, C., and Strosberg, A. D. (1989) Science 245, 1118-1121). 3T3-F442A adipocytes exhibited high and low affinity binding sites for (-)-4-(3-t-butylamino-2-hydroxypropoxy) [5,7-3H]benzimidazole-2-one ((-)-[3H]CGP-12177) (KD = 1.2 and 38.3 nM) and (-)-[125I]iodocyanopindolol ([125I]CYP) (KD = 47 and 1,510 pM). The high affinity sites corresponded to the classical beta 1- and beta 2-AR subtypes whereas the KD values of the low affinity sites for the radioligands were similar to those measured in CHO-beta 3 cells (KD = 28 nM and 1,890 pM for (-)-[3H]CGP12177 and [125I]CYP, respectively). These low affinity sites were undetectable in preadipocytes but represented about 90% of total beta-ARs in adipocytes. The atypical beta-AR and the human beta 3-AR add similarly low affinities (Ki = 3-5 microM) for (+/-)-(2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino-3)-(4-(1-methyl- 4- trifluormethyl-2-imidazolyl)-phenoxy)-2-propanol methane sulfonate (CGP20712A) or erythro-(+/-)-1-(7-methylindan-4-yloxy)-3-isopropylaminob utan-2-ol (ICI118551), highly selective beta 1- and beta 2-AR antagonists, respectively, in agreement with the poor inhibitory effect of the compounds on (-)-isoproterenol (IPR)-stimulated adenylate cyclase activity. Atypical beta-AR and beta 3-AR had an affinity about 10-50 times higher for sodium-4-(2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl)phenoxyace tate sesquihydrate (BRL37344) than the beta 1-AR subtype. This correlates with the potent lipolytic effect of BRL37344 in adipocytes. The rank order of potency of agonists in functional and binding studies was BRL37344 greater than IPR less than (-)-norepinephrine greater than (-)-epinephrine both in 3T3 adipocytes and CHO-beta 3 cells. As in CHO-beta 3 cells, the classical beta 1- and beta 2-antagonists CGP12177, oxprenolol, and pindolol were partial agonists in adipocytes. Although undetectable in preadipocytes, a major mRNA species of 2.3 kilobases (kb) and a minor one of 2.8 kb were observed in adipocytes by hybridization to a human beta 3-AR specific probe.(ABSTRACT TRUNCATED AT 400 WORDS)