Three Generations of ß-blockers: History, Class Differences and Clinical Applicability.

BACKGROUND: Beta-adrenergic receptors are expressed in cardiomyocytes and activated by either noradrenaline released from sympathetic synapses or circulating catecholamines. Their corresponding receptors have three subtypes, namely, ß1, ß2 and ß3, which are members of the G protein-coupled receptors (GPCRs) family. Activation of ß1-adrenergic receptors causes various physiological reactions including cardiac contraction and renin secretion from juxtaglomerular cells of the kidney. Antagonists of ß-adrenergic receptors, known as ß-blockers, have been used effectively for over four decades and have beneficial effects in the treatment of cardiovascular diseases. There are three generations of ß-blockers according to their pharmacological properties. Firstgeneration ß-blockers are non-selective, blocking both ß1- and ß2-receptors; second-generation ß- blockers are more cardioselective in that they are more selective for ß1-receptors; and thirdgeneration ß-blockers are highly selective drugs for ß1-receptors. The latter also display vasodilator actions by blocking α1-adrenoreceptors and activating ß3-adrenergic receptors. In addition, thirdgeneration ß-blockers exhibit angiogenic, antioxidant, anti-proliferative, anti-hypertrophic and antiapoptotic activities among other effects that are still under investigation. CONCLUSION: The objective of this review is to describe the evolution observed during the development of the three distinctive generations, thereby highlighting the advantages of third-generation ß- blockers over the other two drug classes.

Selective ß2-AR Blockage Suppresses Colorectal Cancer Growth Through Regulation of EGFR-Akt/ERK1/2 Signaling, G1-Phase Arrest, and Apoptosis.

The stress-upregulated catecholamines-activated ß1- and ß2-adrenergic receptors (ß1/2-ARs) have been shown to accelerate the progression of cancers such as colorectal cancer (CRC). We investigated the underlying mechanism of the inhibition of ß1/2-ARs signaling for the treatment of CRC and elucidated the significance of ß2-AR expression in CRC in vitro and in clinical samples. The impacts of ß1/2-AR antagonists in CRC in vitro and CRC-xenograft in vivo were examined. We found that repression of ß2-AR but not ß1-AR signaling selectively suppressed cell viability, induced G1-phase cell cycle arrest, caused both intrinsic and extrinsic pathways-mediated apoptosis of specific CRC cells and inhibited CRC-xenograft growth in vivo. Moreover, the expression of ß2-AR was not consistent with the progression of CRC in vitro or in clinical samples. Our data evidence that the expression profiles, signaling, and blockage of ß2-AR have a unique pattern in CRC comparing to other cancers. ß2-AR antagonism selectively suppresses the growth of CRC accompanying active ß2-AR signaling, which potentially carries wild-type KRAS, in vitro and in vivo via the inhibition of ß2-AR transactivated EFGR-Akt/ERK1/2 signaling pathway. Thus, ß2-AR blockage might be a potential therapeutic strategy for combating the progressions of ß2-AR-dependent CRC.

Effects of ß-adrenoceptor subtypes on cardiac function in myocardial infarction rats exposed to fine particulate matter (PM 2.5).

The pathophysiological mechanisms of heart failure (HF) stems were mainly from longstanding overactivation of the sympathetic nervous system and renin-angiotensin-aldosterone system. Recent studies highlighted the potential benefits of ß1-adrenoceptor (ß1-AR) blocker combined with ß2-adrenergic receptor (ß2-AR) agonist in patients with HF. Long-term exposure to fine particulate air pollution, such as particulate matter ≤ 2.5 µm in diameter (PM2.5), has been found associated with acute myocardial infarction (AMI) which is the most common cause of congestive HF. In this study, we have investigated the effect of combined metoprolol and terbutaline on cardiac function in a rat model of AMI exposed to PM2.5. Our results demonstrated that short-term exposure to PM2.5 contributes to aggravate cardiac function in rats with myocardial infarction. The combined use of ß1-AR blocker and ß2-AR agonist is superior to ß1-AR blocker alone for the treatment of AMI rats exposed to PM2.5. The combination of ß1-AR blocker and ß2-AR agonist may decrease the mortality of patients with myocardial infarction who have been exposed to PM2.5.

Beta-adrenergic receptors, from their discovery and characterization through their manipulation to beneficial clinical application.

ß-Adrenergic receptors (ß-AR) are central to the overall regulation of cardiac function. From the first proposed receptor/transmitter concept to the latest clinical ß-blocker trials ß-AR have been shown to play an important role in cardiac disease and heart failure in particular. This study provides a historical perspective, reviews the latest discoveries and beliefs, and discusses the current clinical practices of ß-AR and their modulation with their associated guanine-nucleotide regulatory protein/adenylylcyclasesignal transduction pathways.

ß-Adrenergic receptor subtype signaling in heart: from bench to bedside.

ß-adrenergic receptor (ßAR) stimulation by the sympathetic nervous system or circulating catecholamines is broadly involved in peripheral blood circulation, metabolic regulation, muscle contraction, and central neural activities. In the heart, acute ßAR stimulation serves as the most powerful means to regulate cardiac output in response to a fight-or-flight situation, whereas chronic ßAR stimulation plays an important role in physiological and pathological cardiac remodeling.There are three ßAR subtypes, ß(1)AR, ß(2)AR and ß(3)AR, in cardiac myocytes. Over the past two decades, we systematically investigated the molecular and cellular mechanisms underlying the different even opposite functional roles of ß(1)AR and ß(2)AR subtypes in regulating cardiac structure and function, with keen interest in the development of novel therapies based on our discoveries. We have made three major discoveries, including (1) dual coupling of ß(2)AR to G(s) and G(i) proteins in cardiomyocytes, (2) cardioprotection by ß(2)AR signaling in improving cardiac function and myocyte viability, and (3) PKA-independent, CaMKII-mediated ß(1)AR apoptotic and maladaptive remodeling signaling in the heart. Based on these discoveries and salutary effects of ß(1)AR blockade on patients with heart failure, we envision that activation of ß(2)AR in combination with clinically used ß(1)AR blockade should provide a safer and more effective therapy for the treatment of heart failure.

Noradrenaline acting at central beta-adrenoceptors induces interleukin-10 and suppressor of cytokine signaling-3 expression in rat brain: implications for neurodegeneration.

Evidence indicates that the monoamine neurotransmitter noradrenaline elicits anti-inflammatory actions in the central nervous system (CNS), and consequently may play a neuroprotective role where inflammatory events contribute to CNS pathology. Here we examined the ability of pharmacologically enhancing central noradrenergic tone to induce expression of anti-inflammatory cytokines in rat brain. Administration of the noradrenaline reuptake inhibitor reboxetine (15mg/kg; ip) combined with the alpha(2)-adrenoceptor antagonist idazoxan (1mg/kg; ip) induced interleukin-10 (IL-10) expression in rat cortex and hippocampus. In addition, these drug treatments induced IL-10 signaling as indicated by increased STAT3 phosphorylation and suppressor of cytokine signaling-3 (SOCS-3) mRNA expression. In contrast to the profound increase in IL-10 induced by the reboxetine/idazoxan combination, the other two broad spectrum anti-inflammatory cytokines IL-4 and TGF-beta were not induced by this treatment. The ability of combined treatment with reboxetine and idazoxan to induce IL-10 and SOCS3 expression was mediated by beta-adrenoceptor activation, as their induction was blocked by pre-treatment with the beta-adrenoceptor antagonist propranolol. Moreover, administration of the brain penetrant beta(2)-adrenoceptor agonist clenbuterol induced a time- and dose-dependent increase in central IL-10 and SOCS3 expression, and the ability of clenbuterol to induce IL-10 and SOCS-3 expression was blocked by the centrally acting beta-adrenoceptor antagonist, propranolol, and was mimicked by the highly selective beta(2)-adrenoceptor agonist formoterol. In all, these data indicate that increasing central noradrenergic tone induces IL-10 production and signaling in the CNS, which may protect against neurodegeneration.

Evolving mechanisms of action of beta blockers: focus on nebivolol.

Beta (beta) blockers are widely used for treatment of cardiovascular and noncardiovascular diseases. Nevertheless, their mechanism of action is not fully understood and differs significantly among agents in this class. Chronic increases in adrenergic activity in heart failure result in desensitization of cardiac beta-adrenergic receptor signal transduction and adverse effects on myocytes. By reducing heart rate and decreasing myocardial workload, the pathologic remodeling of the heart may be reversed with beta-blocking agents. Among beta-blockers, there are clear differences in pharmacodynamic and pharmacokinetic properties. Newer beta-blockers differ from older agents with respect to beta-adrenoceptor affinity and selectivity and partial agonist activity, which may affect their mechanism of action and be important in clinical use.The first beta-antagonist compounds were nonselective; the next generation of beta-blockers was selective for beta1-receptors. The most recent beta-blockers may be nonselective or selective, and they have the additional ancillary property of vasodilation. Nebivolol is among the newer third-generation beta-blockers. It is unique in the class, since apart from its cardioselectivity, it also produces nitric oxide-mediated vasodilation. As a result, its hemodynamic profile is clearly different from those of traditional beta-blockers. This review will evaluate this class of agents and the basis for their differences in clinical use.

Gene therapy in heart failure.

With increasing knowledge of basic molecular mechanisms governing the development of heart failure (HF), the possibility of specifically targeting key pathological players is evolving. Technology allowing for efficient in vivo transduction of myocardial tissue with long-term expression of a transgene enables translation of basic mechanistic knowledge into potential gene therapy approaches. Gene therapy in HF is in its infancy clinically with the predominant amount of experience being from animal models. Nevertheless, this challenging and promising field is gaining momentum as recent preclinical studies in larger animals have been carried out and, importantly, there are 2 newly initiated phase I clinical trials for HF gene therapy. To put it simply, 2 parameters are needed for achieving success with HF gene therapy: (1) clearly identified detrimental/beneficial molecular targets; and (2) the means to manipulate these targets at a molecular level in a sufficient number of cardiac cells. However, several obstacles do exist on our way to efficient and safe gene transfer to human myocardium. Some of these obstacles are discussed in this review; however, it primarily focuses on the molecular target systems that have been subjected to intense investigation over the last decade in an attempt to make gene therapy for human HF a reality.

beta-adrenergic relaxation in pulmonary arteries: preservation of the endothelial nitric oxide-dependent beta2 component in pulmonary hypertension.

AIMS: beta-adrenoceptor (beta-AR)-mediated relaxation was characterized in pulmonary arteries from normoxic and hypoxic (as model of pulmonary hypertension) mice. The endothelial NO synthase (eNOS) pathway was especially investigated because of its potential vasculoprotective effects. METHODS: Pulmonary arteries from control or hypoxic (0.5 atm for 21 days) wild-type or eNOS-/- mice were used for pharmacological characterization of beta-AR-mediated relaxation in myograph, and for immunohistochemistry using anti-beta-AR antibodies. RESULTS: In pulmonary arteries from normoxic mice, isoproterenol (beta-AR agonist) and procaterol (selective beta2-AR agonist) elicited relaxation, while cyanopindolol and CL316243 (beta3-AR agonists) were ineffective. The effect of isoproterenol was antagonized by CGP20712A and ICI118551 (beta1- or beta2-AR antagonists, respectively) and also partially inhibited by N omega-nitro-L-arginine methylester (L-NAME, a NOS inhibitor), endothelium denudation, or eNOS gene deletion. Relaxation to procaterol was abolished by L-NAME or endothelium removal. In eNOS-/- mice, procaterol-induced relaxation was decreased but was insensitive to L-NAME, this residual effect involving other endothelium-dependent relaxant factors as compensatory mechanisms. Immunostaining for beta2-AR was observed in the endothelial layer, but not the medial layer of pulmonary arteries. Pulmonary arteries from hypoxic mice exhibited decreased endothelial NO-dependent relaxation to acetylcholine. However, in these arteries, relaxation to procaterol was either unaffected (extralobar segments) or even increased (intralobar segments) and was still abolished by L-NAME or endothelium removal. CONCLUSION: beta1- and beta2-AR, but not beta3-AR, mediate relaxation of mice pulmonary arteries. The beta2-AR component is dependent on eNOS activity and is preserved following chronic hypoxia. These data highlight the role of the beta2-AR as a pharmacological target to induce/restore endothelial NO-dependent protective effects in pulmonary circulation.

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

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

Subclassification and nomenclature of alpha- and beta-adrenoceptors.

The subclassification of alpha- and beta-adrenoceptors has resulted in many opportunities for drug discovery. Important adrenoceptor targets include beta(2)-agonists as bronchodilators, beta(1) or beta(1)/beta(2) antagonists as antihypertensives, centrally acting alpha(2)-agonists for a variety of applications and alpha(1)-antagonists for hypertension and benign prostatic hyperplasia. The pharmacology and nomenclature of 9 adrenoceptors is now established, with alpha(1), alpha(2) and beta-adrenoceptors being divided into three subtypes each. It is unlikely that additional discrete adrenoceptor sequences will be identified; however the presence of "affinity states" can give rise to tissue specific differences in pharmacology for a specific subtype. Polymorphisms and splice variants of adrenoceptors continue to be identified; in some cases these modifications can affect pharmacological characteristics and could influence the efficacy of adrenoceptor-targeted therapy. Selective antagonists are now available of all 9 adrenoceptor subtypes. Although these will not all have therapeutic application, the availability of improved pharmacologic tools could lead to the identification of new adrenoceptor targets.

Recent advances in identification and characterization of beta-adrenoceptor agonists and antagonists.

The three beta-adrenoceptor subtypes (beta(1), beta(2), beta(3)) represent important therapeutic targets. The use of beta(2)-adrenoceptor agonists as bronchodilators and beta(1) or beta(1)/beta(2) antagonists as antihypertensives is well established; research is ongoing in these areas to refine pharmacodynamic properties. It is also feasible to design molecules combining beta-adrenoceptor affinity with other pharmacophores. This is facilitated by the ability to confer beta-adrenoceptor antagonist activity via attachment of a phenylethanolamine moiety or to incorporate diverse structural elements in the N-alkyl substituent of a beta-adrenoceptor agonist or antagonist. beta(3)-Adrenoceptor agonists have not yet been successfully developed as drugs for any therapeutic indication; nevertheless, during the past few years many highly potent and selective beta(3)-agonists have been reported, some with good oral bioavailability. Selective beta(3)-adrenoceptor antagonists have also been identified; useful pharmacological tools are now available for the evaluation of the functional role of each beta-adrenoceptor subtype.

"Phenotypic" pharmacology: the influence of cellular environment on G protein-coupled receptor antagonist and inverse agonist pharmacology.

A central dogma of G protein-coupled receptor (GPCR) pharmacology has been the concept that unlike agonists, antagonist ligands display equivalent affinities for a given receptor, regardless of the cellular environment in which the affinity is assayed. Indeed, the widespread use of antagonist pharmacology in the classification of receptor expression profiles in vivo has relied upon this 'antagonist assumption'. However, emerging evidence suggests that the same gene-product may exhibit different antagonist pharmacological profiles, depending upon the cellular context in which it is expressed-so-called 'phenotypic' profiles. In this commentary, we review the evidence relating to some specific examples, focusing on adrenergic and muscarinic acetylcholine receptor systems, where GPCR antagonist/inverse agonist pharmacology has been demonstrated to be cell- or tissue-dependent, before going on to examine some of the ways in which the cellular environment might modulate receptor pharmacology. In the majority of cases, the cellular factors responsible for generating phenotypic profiles are unknown, but there is substantial evidence that factors, including post-transcriptional modifications, receptor oligomerization and constitutive receptor activity, can influence GPCR pharmacology and these concepts are discussed in relation to antagonist phenotypic profiles. A better molecular understanding of the impact of cell background on GPCR antagonist pharmacology is likely to provide previously unrealized opportunities to achieve greater specificity in new drug discovery candidates.

Functional remodeling in post-myocardial infarcted rats: focus on beta-adrenoceptor subtypes.

Cellular electrophysiological remodeling of the infarcted heart may lead to the deterioration of cardiac function and/or to arrhythmias. The present study was designed to characterize the functional expression of the hyperpolarization-activated current (I(f)) and its modulation by beta(1)-, beta(2)- and beta(3)-adrenoceptor (AR) subtypes, in patch-clamped ventricular myocytes isolated from the heart of post-myocardial infarcted (PMI) rats and sham-operated control (SHAM) rats. Maximum specific conductance of I(f) was significantly higher in left ventricular myocytes (LVM) from PMI rats compared to right ventricular myocytes from PMI rats as well as LVM and RVM from SHAM rats. All other basic properties of I(f) were similar. beta(1)AR stimulation with noradrenaline caused a rightward shift of V(H) in LVM from PMI rats which was significantly smaller (52.2%) than in LVM from SHAM rats. Incubation with pertussis toxin (PTX) largely restored the effect of beta(1)AR in PMI cells (86.6% vs. SHAM cells), but did not affect beta(1)AR response in SHAM cells. beta(2)AR response was significantly and equally increased by PTX-pretreatment (by 94% in SHAM and 87% in PMI cells). Conversely, beta(3)AR stimulation by the selective agonist SR 58611A caused a leftward shift of the activation curve which was significantly larger in PMI cells than in SHAM cells (P<0.01). beta(3)AR response was blunted by PTX-pretreatment, by incubation with N(G)-monomethyl-l-arginine acetate or by the selective beta(3)AR antagonist SR 59230A 1 microM. In conclusion, I(f) is significantly overexpressed in LVM from PMI rat hearts. In these cells, I(f) modulation by beta(1)AR is significantly depressed while beta(3)AR modulation is markedly enhanced, probably reflecting the increased activity of PTX-sensitive G(i) proteins in PMI cells.

The potency of KUL-7211, a selective ureteral relaxant, in isolated canine ureter: comparison with various spasmolytics.

We compared the potency of a selective ureteral relaxant KUL-7211 (beta(2)/beta(3)-adrenoceptor agonist; (-)-2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenyloxy]acetic acid) with those of various spasmolytics on contractions in isolated canine ureteral preparations. Drug effects were evaluated on the tonic contraction induced by KCl (80 mM) and on spontaneous, 1x10(-5) M phenylephrine-, and 1x10(-6) M PGF(2alpha)-induced rhythmic contractions in isolated canine ureteral preparations using a functional experimental technique. The potencies (pD(2) value) of the following drugs were compared: KUL-7211, tamsulosin (an alpha(1A/1D)-adrenoceptor antagonist), prazosin (an alpha(1)-adrenoceptor antagonist), verapamil (a Ca(2+)-channel blocker), butylscopolamine (a nonselective muscarinic antagonist), and papaverine (a phosphodiesterase inhibitor). The rank order of relaxing potencies against KCl-induced tonic contraction was KUL-7211 (6.60)>tamsulosin(5.90)>verapamil(5.70)>papaverine(4.88)>prazosin (4.54). The rank order of potencies for reductions in spontaneous rhythmic contractions was KUL-7211 (6.80)>verapamil(6.12)>papaverine(5.05). Conversely, high concentrations of the two alpha-adrenoceptor antagonists (tamsulosin and prazosin) and of butylscopolamine enhanced the spontaneous contractions, although at low concentrations (up to 1x10(-6) M) they had no significant effects. For suppression of spasmogen-induced rhythmic contractions, the rank order of potencies was, against phenylephrine-induced contractions: KUL-7211 (6.95)>tamsulosin(6.26)>prazosin(5.68)>verapamil(5.64)>papaverine (5.03), and against PGF(2alpha)-induced contractions: KUL-7211 (7.05)>verapamil(6.70)>papaverine (5.27). Our results suggest that in dogs, the beta(2)/beta(3)-adrenoceptor agonist KUL-7211 is the most efficacious ureteral relaxant among the spasmolytics tested against various contractions. Possibly, KUL-7211 might be useful for promoting stone passage and relieving ureteral colic in urolithiasis patients.

New insights into beta-adrenoceptors in smooth muscle: distribution of receptor subtypes and molecular mechanisms triggering muscle relaxation.

1. The beta-adrenoceptor is currently classified into beta(1), beta(2) and beta(3) subtypes and all three subtypes are expressed in smooth muscle. Each beta-adrenoceptor subtype exhibits tissue-specific distribution patterns, which may be a determinant controlling the mechanical functions of corresponding smooth muscle. Airway and uterine smooth muscles abundantly express the beta(2)-adrenoceptor, the physiological significance of which is established as a fundamental regulator of the mechanical activities of these muscles. Recent pharmacomechanical and molecular approaches have revealed roles for the beta(3)-adrenoceptor in the gastrointestinal tract and urinary bladder smooth muscle. 2. The beta-adrenoceptor is a G(s)-protein-coupled receptor and its activation elevates smooth muscle cAMP. A substantial role for a cAMP-dependent mechanism(s) is generally believed to be the key trigger for eliciting beta-adrenoceptor-mediated relaxation of smooth muscle. Downstream effectors activated via a cAMP-dependent mechanism(s) include plasma membrane K(+) channels, such as the large-conductance, Ca(2+)-activated K(+) (MaxiK) channel. 3. Beta-Adrenoceptor-mediated relaxant mechanisms also include cAMP-independent signalling pathways. This view is supported by numerous pharmacological and electrophysiological lines of evidence. In airway smooth muscle, direct activation of the MaxiK channel by G(s)alpha is a mechanism by which stimulation of beta(2)-adrenoceptors elicits muscle relaxation independently of the elevation of cAMP. 4. The cAMP-independent mechanism(s) is also substantial in beta(3)-adrenoceptor-mediated relaxation of gastrointestinal tract smooth muscle. However, in the case of the beta(3)-adrenoceptor, a delayed rectified K(+) channel rather than the MaxiK channel seems to mediate, in part, cAMP-independent relaxant mechanisms. 5. In the present article, we review the distribution of beta-adrenoceptor subtypes in smooth muscle tissues and discuss the molecular mechanisms by which each subtype elicits muscle relaxation, focusing on the roles of cAMP and plasma membrane K(+) channels.

Norepinephrine induces lipolysis in beta1/beta2/beta3-adrenoceptor knockout mice.

Catecholamines are major stimulants of adipose tissue metabolism. Norepinephrine and epinephrine act through three subtypes of beta-adrenoceptors (beta-AR) expressed in the adipocytes. The aim of this work was to study the mechanisms of lipid mobilization in beta1/beta2/beta3-AR triple-knockout (beta-less) mice. Glycerol and nonesterified fatty acids released from isolated adipocytes were measured as an index of lipolytic activity. There was no difference between the two genotypes for basal lipolysis and lipolytic response to corticotropin or to agents acting at the adenylyl cyclase and protein kinase A levels. The lipolytic response to norepinephrine and beta-AR agonists was blunted in beta-less mice. However, a residual low-affinity lipolytic effect was observed in the presence of catecholamines and beta3-AR agonists but not of beta1- or beta2-AR agonists. cAMP levels were increased by a beta-AR agonist in white and brown adipocytes of beta-less mice. The residual lipolytic effect was blocked by beta-AR antagonists. It was mediated neither by alpha1- or alpha2-AR nor dopaminergic, serotonergic, and histaminergic by receptors. Bioinformatic analyses do not provide evidence for a fourth beta-AR. We conclude that the residual lipolytic effect observed in beta-less mice can be attributed to an unknown Gs-protein-coupled receptor with low affinity for catecholamines.

Functional characterization of the beta-adrenergic receptor subtypes expressed by CA1 pyramidal cells in the rat hippocampus.

Recent studies have demonstrated that activation of the beta-adrenergic receptor (AR) using the selective beta-AR agonist isoproterenol (ISO) facilitates pyramidal cell long-term potentiation in the cornu ammonis 1 (CA1) region of the rat hippocampus. We have previously analyzed beta-AR genomic expression patterns of 17 CA1 pyramidal cells using single cell reverse transcription-polymerase chain reaction, demonstrating that all samples expressed the beta2-AR transcript, with four of the 17 cells additionally expressing mRNA for the beta1-AR subtype. However, it has not been determined which beta-AR subtypes are functionally expressed in CA1 for these same pyramidal neurons. Using cell-attached recordings, we tested the ability of ISO to increase pyramidal cell action potential (AP) frequency in the presence of subtype-selective beta-AR antagonists. ICI-118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] and butoxamine [alpha-[1-(t-butylamino)ethyl]-2,5-dimethoxybenzyl alcohol) hydrochloride], agents that selectively block the beta2-AR, produced significant parallel rightward shifts in the concentration-response curves for ISO. From these curves, apparent equilibrium dissociation constant (K(b)) values of 0.3 nM for ICI-118,551 and 355 nM for butoxamine were calculated using Schild regression analysis. Conversely, effective concentrations of the selective beta1-AR antagonists CGP 20712A [(+/-)-2-hydroxy-5-[2-([2-hydroxy-3-(4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy)propyl]amino)ethoxy]-benzamide methanesulfonate] and atenolol [4-[2'-hydroxy-3'-(isopropyl-amino)propoxy]phenylacetamide] did not significantly affect the pyramidal cell response to ISO. However, at higher concentrations, atenolol significantly decreased the potency for ISO-mediated AP frequencies. From these curves, an apparent atenolol K(b) value of 3162 nM was calculated. This pharmacological profile for subtype-selective beta-AR antagonists indicates that beta2-AR activation is mediating the increased AP frequency. Knowledge of functional AR expression in CA1 pyramidal neurons will aid future long-term potentiation studies by allowing selective manipulation of specific beta-AR subtypes.