From the Cover: β2-Adrenoceptor signaling is required for the development of an asthma phenotype in a murine model

Chronic regular use of β₂-adrenoceptor (β₂-AR) agonists in asthma is associated with a loss of disease control and increased risk of death. Conversely, we have found that administration of β₂-AR inverse agonists results in attenuation of the asthma phenotype in an allergen-driven murine model. Besides antagonizing agonist-induced signaling and reducing signaling by empty receptors, β-AR inverse agonists can also activate signaling by novel pathways. To determine the mechanism of the β-AR inverse agonists, we compared the asthma phenotype in β₂-AR-null and wild-type mice. Antigen challenge of β₂-AR-null mice produced results similar to what was observed with chronic β₂-AR inverse agonist treatment, namely, reductions in mucous metaplasia, airway hyperresponsiveness (AHR), and inflammatory cells in the lungs. These results indicate that the effects of β₂-AR inverse agonists are caused by inhibition of β₂-AR signaling rather than by the induction of novel signaling pathways. Chronic administration of alprenolol, a β-blocker without inverse agonist properties, did not attenuate the asthma phenotype, suggesting that it is signaling by empty receptors, rather than agonist-induced β₂-AR signaling, that supports the asthma phenotype. In conclusion, our results demonstrate that, in a murine model of asthma, β₂-AR signaling is required for the full development of three cardinal features of asthma: mucous metaplasia, AHR, and the presence of inflammatory cells in the lungs.     

Association between nonspecific airway hyperresponsiveness and Arg16Gly beta2-adrenergic receptor gene polymorphism in asymptomatic healthy Japanese subjects

BACKGROUND: Nonspecific airway hyperresponsiveness (AHR), a cardinal feature of asthma, is thought to result from several genetic and environmental factors. Asymptomatic AHR in nonasthmatic healthy subjects might be a risk factor for the development of asthma. Genetic variations in codons 16 and 27 of the human beta(2)-adrenergic receptor (beta(2)-AR) alter receptor function in vitro and are associated with various asthma-related phenotypes, including asthma severity and AHR. To date, however, few reports have examined the impact of beta(2)-AR gene polymorphism on AHR in asymptomatic healthy subjects. OBJECTIVE: To determine whether polymorphism of the beta(2)-AR gene (Arg16Gly and Gln27Glu) might influence nonspecific AHR in asymptomatic healthy Japanese subjects. DESIGN AND PARTICIPANTS: A cohort of 120 asymptomatic healthy subjects was analyzed using a stepwise linear regression model. Nonspecific airway responsiveness was measured using a continuous methacholine inhalation method (Astograph; Chest; Tokyo, Japan). We used values of the cumulative dose of inhaled methacholine measured at the inflection point at which respiratory conductance starts to decrease (Dmin) as an index of AHR. Genotyping to identify polymorphisms at codons 16 and 27 was conducted using an assay combining kinetic real-time quantitative polymerase chain reaction with allele-specific amplification. RESULTS: The Gly16Gly genotype was associated with lower Dmin values. The log Dmin value of asymptomatic healthy subjects carrying the Arg16 allele (Arg16/Arg or Arg16/Gly, n = 90) was 1.09 +/- 0.56 (mean +/- SD), while those homozygous for the Gly16 allele (n = 30) yielded a log Dmin value of 0.85 +/- 0.56 (p < 0.05). CONCLUSION: This study indicates that a specific beta(2)-AR polymorphism at codon 16 might be a genetic determinant of AHR, as judged by methacholine-induced bronchoconstriction in asymptomatic healthy subjects.     

Beta-adrenergic receptor blockade modulates Bcl-X(S) expression and reduces apoptosis in failing myocardium

The mechanisms by which beta-adrenergic receptor (beta-AR) blockade modulates apoptosis in heart failure (HF) are unclear. We examined the impact of beta-AR blockade with metoprolol on myocardial remodeling, apoptosis, pro-apoptotic (Fas, Fas ligand, Bax, and Bcl-X(S)) and anti-apoptotic (Bcl-X(L)and Bcl-2) gene expression, and Bcl-X(L) and Bcl-X(S) protein in post-infarction HF in rats. In untreated rats, there was significant (P < 0.001) LV dilatation and systolic dysfunction compared to sham. Myocardial apoptosis was significantly increased (P < 0.005). Fas, Bax, and Bcl-2 mRNA expression was unchanged. However, Fas ligand mRNA and Bcl-X(S) mRNA and protein, all undetectable in sham, were markedly elevated (P < 0.001), whereas Bcl-X(L) mRNA and protein was unchanged. Immunohistochemistry confirmed increased Bcl-X(S) staining in failing myocardium, with unchanged Bcl-X(L). Metoprolol treatment resulted in: (1) improved LV remodeling (P < 0.025), (2) reduced myocardial apoptosis (P < 0.005), and (3) selective reduction in myocardial Bcl-X(S) expression (P < 0.001) without change in Fas, Fas ligand, Bax, Bcl-2, or Bcl-X(L). Studies in isolated rat myocytes revealed that prolonged isoproterenol (ISO) stimulation significantly increased Bcl-X(S) protein, reducing the Bcl-X(L)/X(S) ratio and myocyte survival (P < 0.005). ISO-induced Bcl-X(S) expression was significantly attenuated (P < 0.001) by both metoprolol and CGP20712A, a beta1-AR selective antagonist, but not by ICI118,551, a beta2-AR selective antagonist. We conclude that adrenergic activation, such as occurs in HF, increases pro-apoptotic Bcl-X(S) expression via the beta1-AR. beta-AR blockade in HF reduces myocardial apoptosis; attenuation of Bcl-X(S) expression may be one mechanism underlying this effect.     

Canine Ventricular Myocyte β2-Adrenoceptors Are Not Functionally Coupled to L-Type Calcium Current

INTRODUCTION: To establish the functional coupling of beta adrenoceptor (betaAR) subtypes beta1AR and beta2AR to L-type calcium current (I(CaL)), we investigated the nonselective agonist isoproterenol (ISO) and the relatively selective beta2AR agonists zinterol (ZIN) and salbutamol (SAL) on I(CaL) in isolated canine ventricular myocytes in the presence and absence of CGP 20712A (CGP) and atenolol (AT), selective beta1AR antagonists, and ICI 118,551 (ICI) a selective beta2AR antagonist. METHODS AND RESULTS: Peak I(CaL) was determined using "patch type" microelectrodes and whole cell voltage clamp. ISO (0.5 microM) increased I(CaL) maximally 3.5 +/- 0.67 fold. ZIN (10.0 microM) and SAL (10.0 microM) increased I(CaL) maximally 1.5 +/- 0.2 fold (n = 5) and 1.4 +/- 0.1 fold (n = 5), respectively. These effects were fully inhibited by CGP (0.3 microM) and AT (1.0 microM), which are inhibitors of beta1AR, but not by ICI (0.1 microM), which is a beta2AR inhibitor. ZIN at relatively lower concentrations (< or = 0.1 microM) did not increase I(CaL). CGP (0.3 microM) but not AT and ICI inhibited I(CaL) in the absence of betaAR agonists. CGP inhibition of I(CaL) was absent in the presence of forskolin (1.0 microM), which increases cAMP levels and I(CaL) by directly stimulating the adenylate cyclase. These data indicate that none of the antagonists affect I(CaL) through an action downstream of betaAR. CONCLUSION: Beta-adrenergic agonists increase I(CaL) via beta1AR but not beta2AR in canine ventricular myocytes.     

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.     

An increase in murine skeletal muscle peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) mRNA in response to exercise is mediated by beta-adrenergic receptor activation

A single bout of exercise increases expression of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha mRNA, which may promote mitochondrial biogenesis in skeletal muscle. In brown adipose tissue, cold exposure up-regulates PGC-1alpha expression via adrenergic receptor (AR) activation. Because exercise also activates the sympathetic nervous system, we examined whether exercise-induced increase in PGC-1alpha mRNA expression in skeletal muscle was mediated via AR activation. In C57BL/6J mice, injection of the beta2-AR agonist clenbuterol, but not alpha-, beta1-, or beta3-AR agonists, increased PGC-1alpha mRNA expression more than 30-fold in skeletal muscle. The clenbuterol-induced increase in PGC-1alpha mRNA expression in mice was inhibited by pretreatment with the beta-AR antagonist propranolol. In ex vivo experiments, direct exposure of rat epitrochlearis to beta2-AR agonist, but not alpha-, beta1-, and beta3-AR agonist, led to an increase in levels of PGC-1alpha mRNA. Injection of beta2-AR agonist did not increase PGC-1alpha mRNA expression in beta1-, beta2-, and beta3-AR knockout mice (beta-less mice). PGC-1alpha mRNA in gastrocnemius was increased 3.5-fold in response to running on a treadmill for 45 min. The exercise-induced increase in PGC-1alpha mRNA was inhibited by approximately 70% by propranolol or the beta2-AR-specific inhibitor ICI 118,551. The exercise-induced increase in PGC-1alpha mRNA in beta-less mice was also 36% lower than that in wild-type mice. These data indicate that up-regulation of PGC-1alpha expression in skeletal muscle by exercise is mediated, at least in part, by beta-ARs activation. Among ARs, beta2-AR may mediate an increase in PGC-1alpha by exercise.     

Role of nitric oxide/cyclic GMP and cyclic AMP in beta3 adrenoceptor-chronotropic response

In this study we determine different signaling pathways involved in beta(3) adrenoceptor (beta(3)-AR) dependent frequency stimulation in isolated rodent atria. Promiscuous coupling between different G-proteins and beta(3)-AR could explain the multiple functional effects of beta(3)-AR stimulation. We examine the mechanisms and functional consequences of dual adenylate cyclase and guanylate cyclase pathways coupling to beta(3)-AR in isolated rodent atria. The beta(3)-AR selective agonists ZD 7114 and ICI 215001 stimulated in a dose-dependent manner the contraction frequency that significantly correlated with cyclic AMP (cAMP) accumulation. Inhibition of adenylate cyclase shifted the chronotropic effect to the right. On the other hand, the ZD 7114 activity on frequency was enhanced by the inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase. This countervailing negative chronotropic nitric oxide-cyclic GMP (NO-cGMP) significantly correlated with the increase on NOS activity and cGMP accumulation. Current analysis showed a negative cross talk between cAMP chronotropic and NO-cGMP effects by inhibition of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), NOS isoforms and Gi-protein on the effects of beta(3)-AR stimulation. RT-PCR detected both eNOS and nNOS in isolated rat atria. NOS isoforms performed independently. Only nNOS participated in limiting the effect of beta(3)-AR stimulation. In eNOS-KO (eNOS-/-) mice the chronotropic effect of beta(3)-AR agonists did not differ from wild type (WT) mice atria, but it was increased by the inhibition of nNOS activity. Our results suggest that the increase in frequency by beta(3)-AR activation on isolated rodent atria is associated to a parallel increases in cAMP. The nNOS-cGMP pathway negatively modulates beta(3)-AR activation. Multiple signal transduction pathways between G-protein and beta(3)-AR may protect myocardium from catecholamine-induced cardiotoxic effects.     

大鼠心肌梗死后心肌组织β_2受体表达水平及其对受体后信号的作用

目的:研究大鼠心肌梗死后心肌组织β_2肾上腺素受体(β_2受体)mRNA表达水平及β_2受体对细胞受体后信号3',5'-环化一磷酸腺苷酸(cAMP)含量和cAMP依赖蛋白激酶(PKA)活性作用的动态变化.方法:Wistar大鼠48只制备心肌梗死模型,随机分为梗死后2周组、4周组、8周组,另制备假手术模型为假手术组.分离心肌细胞.每毫升细胞悬液作为一份样品,按给药不同随机分为7类:①空白对照;②沙丁胺醇;③沙丁胺醇+百日咳毒素;④异丙肾上腺素;⑤异丙肾上腺素+ICI118,551(β_2受体拮抗剂);⑥异丙肾上腺素+阿替洛尔;⑦异丙肾上腺素+普萘洛尔,每类10份样品,测定cAMP含量和PKA活性.以逆转录多聚酶链反应方法测定β_2受体、β_1受体mRNA表达水平.结果:心肌梗死后β_1受体mRNA表达水平逐渐下降(P<0.05),β_2受体mRNA水平在β受体中所占比例由19%升高至38%(P<0.01),差异有统计学意义;与空白对照比较,在假手术组和梗死后2周组、梗死后4周组、梗死后8周组心肌细胞,沙丁胺醇可使cAMP含量分别升高98.1%、133.6%、147.7%和150.7%(P均<0.05),差异有统计学意义.与异丙肾上腺素比较,ICI118,551仅在梗死后8周组心肌细胞可抑制异丙肾上腺素升高cAMP含量的作用(P<0.05);阿替洛尔在假手术组和梗死后2周组、梗死后4周组细胞可抑制此作用(P<0.05);普萘洛尔在4组均可抑制此作用(P<0.05).ICI118,551仅在梗死后8周组心肌细胞可抑制异丙肾上腺素升高依赖蛋白激酶活性的作用达55.80%(P<0.05);阿替洛尔仅在假手术组和梗死后2周组心肌细胞可抑制此作用,分别为44.76%、34.59%(P均<0.05);普萘洛尔在假手术组、梗死后2周组、梗死后4周组、梗死后8周组心肌细胞均可抑制此作用,分别为49.60%、40.82%、40.64%、46.84(P均<0.05),差异有统计学意义.结论:心肌梗死后β_2受体mRNA水平在β受体中所占比例升高.β_2受体激动显著升高心肌细胞cAMP含量和cAMP依赖蛋白激酶活性(P<0.05),对心肌梗死后心肌细胞的作用较正常心肌细胞增强.     

Electrophysiological characterization of cardiac beta 2-adrenoceptors in sheep Purkinje fibers

The electrophysiological effects mediated by beta 2-adrenoceptor stimulation were studied in sheep cardiac Purkinje fibers. beta 2-Adrenoceptor stimulation was achieved by using isoproterenol (ISO) in the presence of the highly selective beta 1-antagonist CGP 20712A. ICI 118,551 was used as a selective beta 2-antagonist. In driven preparations, ISO (0.1 to 1 microM) caused a positive inotropic effect which was associated with the shortening of action potential duration and was completely abolished only by the simultaneous presence of CGP 20712A (0.3 microM) and ICI 118,551 (50 nM). In previously quiescent preparations, ISO (0.1 to 1 microM) induced spontaneous activity in 15 out of 24 preparations. In the presence of CGP 20712A (0.3 microM) only five preparations out of 24 became automatic when exposed to ISO, and their rate of firing was significantly reduced (13 +/- 4 vs. 43 +/- 6 beats/min, P less than 0.05) with respect to ISO alone. CGP 20712A completely abolished the steepening of diastolic depolarization and the increase of the pacemaker current caused by ISO (0.1 to 1 microM). The beta 2-antagonist ICI 118,551 (50 nM) completely failed to modify the effect of ISO on the rate of spontaneous firing, the slope of diastolic depolarization and the pacemaker current. On the other hand, the increase of oscillatory afterpotential (OAP) amplitude caused by ISO in strophanthidin-treated preparations was significantly reduced only by the beta 1-antagonist CGP 20712A, but not by the beta 2-antagonist ICI 118,551. These results demonstrate that beta 2-adrenoceptors are functionally present in sheep Purkinje fibers where their stimulation consistently causes a positive inotropic effect. However, beta 2-adrenoceptors do not appear to affect the processes of normal automaticity, and do not greatly contribute to triggered activity due to OAPs.     

Evidence for the existence of postsynaptic beta-1 and beta-2 adrenoceptors in isolated simian facial veins

Summary With the use of a steel cannula inserting method, the actions of the beta-adrenoceptor agonists, noradrenaline (NA, a mixed agonist), isoprenaline (a mixed agonist), dobutamine (a selective beta-1 agonist), salbutamol, and procaterol (selective beta-2 agonists), were investigated on isolated and perfused simian facial veins. Each beta-agonist usually induced a vasodilation in a dose-related manner in non-preconstricted vessel preparations. The rank order of potency was isoprenaline NA > dobutamine > salbutamol > procaterol. NA- and isoprenaline-induced vasodilations were inhibited by either metoprolol (a selective beta-1 adrenoceptor antagonist) or ICI 118,551 (a selective beta-2 antagonist). After beta-1 blockade, NA produced a vasoconstriction which was readily blocked by bunazosin (an alpha-1 antagonist). Dobutamine-induced vasodilations were strongly suppressed by metoprolol and slightly blocked by ICI 118,551. Salbutamol-induced vasodilations were blocked by metoprolol, while ICI 118,551 more markedly inhibited these dilations. From these results, it was concluded that there are abundant beta-adrenoceptors and predominantly beta-1 adrenoceptors in isolated simian facial veins.     

Pharmacological modulation of the hyperpolarization-activated current (I f) in human atrial myocytes: focus on G protein-coupled receptors

AIMS: In human atrial myocytes (HuAM) two beta-adrenergic receptors (beta-AR) and four splicing-variants of the serotonin 5-HT(4) receptor are present. Multiple coupling with G stimulatory (G(s)) and G inhibitory (G(i)) proteins has been proposed for both beta(2)-AR and 5-HT((4b)) subtypes, but no functional data exist in HuAM. Serotonin (5-HT) and catecholamines are able to trigger arrhythmias in human atrium, but the underlying cellular mechanisms are not completely understood. The pacemaker current (I(f)) is an inward Na(+)/K(+) current, constitutively present in HuAM and directly modulated by cAMP; I(f) could play a role in triggering human atrial arrhythmias. This study evaluated the different G protein coupling of beta(1)-AR, beta(2)-AR and 5-HT(4) receptors by assessing the modulation of I(f) by selective stimuli. METHODS: HuAM were isolated from right atrial appendages and utilized for patch-clamp recording. The coupling of receptor subtypes with G(i) proteins was tested by incubating HuAM in pertussis toxin (PTX). RESULTS: Beta(1)-AR stimulation (Isoprenaline [ISO] + ICI 118,551), and 5-HT caused a concentration-dependent significant shift of the half activation potential of I(f) activation curve (DeltaV(h)), P < 0.01. beta(2)-AR stimulation (ISO 1 microM + CGP 20712A) also significantly shifted V(h) (P < 0.0001), but with DeltaV(h)[beta(2)-AR] significantly smaller than the effect caused by 1 microM beta(1)-AR stimulation (P < 0.05). Pre-treatment of HuAM with PTX did not alter the effect of beta(1)-AR stimulation (both 0.1 and 1 microM) and 1 microM 5-HT on I(f), but significantly increased the effect in response to beta(2)-AR stimulation and 0.1 microM 5-HT (P < 0.05 for both), thus suggesting a G(i) protein coupling of these receptors. CONCLUSIONS: Our results provide the first functional evidence of the different G protein coupling of beta(1)-AR, beta(2)-AR and 5-HT(4) receptors in HuAM. Further they support the view that I(f) current might play an important role in triggering catecholamines and serotonin-induced atrial arrhythmias.     

The role of the hypothalamic beta-adrenergic system in controlling the LH rise in short-term castrated rats

Intraventricular infusions of adrenaline and various pharmacological agents acting on beta-adrenergic receptor subtypes were carried out in rats orchidectomized 16 h previously. Infusions (10 microliter) of solutions containing the drugs were administered under anaesthesia induced with alphaxalone and alphadolone. Levels of LH were measured in plasma collected immediately before and at predetermined intervals after the infusion. The acute rise in LH levels after castration was increased still further by isoprenaline (a mixed beta 1- and beta 2-agonist), fenoterol (a beta 2-agonist) and atenolol (a beta 1-antagonist). In contrast, prenalterol (a beta 1-agonist) and (2RS,3RS)-3-isopropylamino-1-(7-methylindan-4-yloxy)++ +butan-2-ol (ICI 118,551) (a selective beta 2-antagonist) were inhibitory to LH release. Adrenaline itself, salbutamol (another selective beta 2-agonist), propranolol (a mixed beta-antagonist) and metoprolol (a beta 1-antagonist) did not significantly alter plasma LH concentrations at the doses administered. The stimulatory effect of isoprenaline on LH release was partially reduced when given together with ICI 118,551, but was not affected when administered simultaneously with atenolol. The inhibitory effect of ICI 118,551 was, however, prevented by concomitant administration with fenoterol, as was that of prenalterol when infused with atenolol. The results suggest that the hypothalamic mediation of the short-term changes in LH release in response to castration is exerted, at least in part, through the activation of a beta 2-stimulatory component and the suppression of a beta 1-inhibitory component.     

Influence of beta 2-adrenoceptor stimulation and blockade on cardiac electrophysiologic properties and serum potassium concentration in the anesthetized dog

The influence of selective beta 2-adrenoceptor stimulation and blockade on cardiac electrophysiologic properties was studied in 18 anesthetized dogs. Selective beta 2-adrenoceptor activation by salbutamol failed to alter myocardia excitability but significantly lowered serum potassium concentration. Excitability, refractoriness, and ventricular fibrillation threshold were also not changed after administration of 100 and 200 micrograms/kg of the selective beta 2-antagonist ICI 118,551. However, at a dose of 500 micrograms/kg, refractoriness was prolonged and ventricular fibrillation threshold increased. These changes appear to be due to blockade of beta 1-adrenoceptors rather than to membrane stabilizing effects, since in catecholamine-depleted animals even the highest dose of ICI 118,551 did not alter myocardial electrical properties. It is concluded that beta 2-adrenoceptors do not influence electrophysiologic properties of the canine myocardium.     

Development of beta 3-adrenoceptor agonists for the treatment of obesity and diabetes--an update.

Beta 3-adrenoceptor (beta 3-AR) agonists were found to have remarkable anti-obesity and anti-diabetic effects in rodents shortly after their discovery in the early 1980s. Despite these promising qualities, several pharmaceutical problems and theoretical concerns have slowed the development of these products as therapeutic agents in humans during the last 15 years. To date, the pharmaceutical industry has not been successful in developing a beta 3-AR agonist for use in the treatment of human obesity and type 2 diabetes. Pharmaceutical problems in this area concern important differences between rodent and human beta 3-AR and the difficulty in finding a compound with sufficient bioavailability that is a highly selective and full agonist at the human receptor. Some of these problems seem to have been solved with the cloning of the human beta 3-AR, which has made it possible to develop novel compounds directly and specifically against the human receptor. However, several theoretical concerns still remain. These include the major question as to whether the number of biologically active beta 3-ARs in adult humans is sufficient to produce relevant metabolic effects and, if so, whether their long-term stimulation is safe and free of unwarranted side effects. In addition, the mechanisms of action of beta 3-AR agonists remain poorly understood. Recent studies using CL 316,243, a highly selective beta 3-adrenergic compound, have provided new insights into the potential mechanisms of action of these drugs in rodents as well as the first evidence that treatment with a highly selective beta 3-AR agonist exerts relevant metabolic effects in humans. It appears that chronic beta 3-adrenergic stimulation in white adipose tissue increases the expression of newly discovered mitochondrial uncoupling proteins (UCP 2 and 3) and a "reawakening" of dormant brown adipocytes. In addition, beta 3-ARs may be present in skeletal muscle where ectopic expression of UCP-1 has been reported. If these findings are confirmed, tissues other than brown fat may play an important role in mediating beta 3-adrenergic effects on thermogenesis and substrate oxidation. In humans, treatment with CL 316,243 for 8 weeks, in spite of limited bioavailability, induced marked plasma concentration-dependent increases in insulin sensitivity, lipolysis, and fat oxidation in lean volunteers, without causing beta 1-, or beta 2-mediated side effects. These results clearly indicate that favourable metabolic effects can be achieved by selective beta 3-AR stimulation in humans. The compounds of the next generation currently emerging from preclinical development are full agonists at the human beta 3-AR. These agents have demonstrated promising results in non-human primates. It will be interesting to see whether their efficacy in clinical trials is superior to that achieved with previous (rodent) beta 3-AR agonists and, if so, whether their effects will eventually translate into weight loss and improved metabolic control that could facilitate their use as effective drugs for the treatment of obesity and Type 2 diabetes in humans.     

Noradrenergic regulation of growth hormone secretion in the baboon

We have investigated the effects of iv administered noradrenergic agonists and antagonists on plasma GH concentration in the adolescent baboon, Papio papio, with the aim of defining the relative roles of adrenergic receptor subtypes (alpha 1, alpha 2, beta 1, and beta 2) in the regulation of GH release. Clonidine (0.02 mg/kg) or UK-14,304 (0.02 mg/kg), potent centrally acting alpha 2 noradrenergic agonists, were infused into 24 animals pretreated with either saline, or selective alpha 1 and alpha 2 noradrenergic antagonists. Both agonists potently augment plasma GH, producing peak levels of 30-60 ng/ml 15 min post infusion. These responses can be prevented by the prior infusion of the alpha 2 antagonist, piperoxane (1.0 mg/kg), but not by the alpha 1 antagonist, prazosin (2.0 mg/kg). Log dose response curves of the 2 agonists demonstrate a greater potency for UK-14,304 vs. clonidine on a molar basis. In animals pretreated with monoamine depleting agents (reserpine and alpha-methyl paratyrosine) the plasma GH response to an infusion of clonidine (0.02 mg/kg) is significantly enhanced (P less than 0.001). Beta-Adrenoreceptor antagonism by propranolol (0.02 or 1.0 mg/kg) or the more selective beta 2-adrenoreceptor antagonist, ICI 118,551 (0.02-1.0 mg/kg), results in a rapid and significant (P less than 0.01) increase in plasma GH. The beta 1-antagonist, practolol (0.2-2.0 mg/kg), does not alter plasma GH levels. It is proposed that in the baboon, noradrenaline acts on alpha 2-noradrenergic receptors to stimulate GH release and on beta 2-noradrenergic receptors to inhibit GH release.     

The beta(2)-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through G(i)-dependent coupling to phosphatidylinositol 3'-kinase

Recent studies have shown that chronic beta-adrenergic receptor (beta-AR) stimulation alters cardiac myocyte survival in a receptor subtype-specific manner. We examined the effect of selective beta(1)- and beta(2)-AR subtype stimulation on apoptosis induced by hypoxia or H(2)O(2) in rat neonatal cardiac myocytes. Although neither beta(1)- nor beta(2)-AR stimulation had any significant effect on the basal level of apoptosis, selective beta(2)-AR stimulation protected myocytes from apoptosis. beta(2)-AR stimulation markedly increased mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) activation as well as phosphatidylinositol-3'-kinase (PI-3K) activity and Akt/protein kinase B phosphorylation. beta(1)-AR stimulation also markedly increased MAPK/ERK activation but only minimally activated PI-3K and Akt. Pretreatment with pertussis toxin blocked beta(2)-AR-mediated protection from apoptosis as well as the beta(2)-AR-stimulated changes in MAPK/ERK, PI-3K, and Akt/protein kinase B. The selective PI-3K inhibitor, LY 294002, also blocked beta(2)-AR-mediated protection, whereas inhibition of MAPK/ERK activation at an inhibitor concentration that blocked agonist-induced activation but not the basal level of activation had no effect on beta(2)-AR-mediated protection. These findings demonstrate that beta(2)-ARs activate a PI-3K-dependent, pertussis toxin-sensitive signaling pathway in cardiac myocytes that is required for protection from apoptosis-inducing stimuli often associated with ischemic stress.     

Sepsis is associated with an upregulation of functional beta3 adrenoceptors in the myocardium

OBJECTIVE: To analyze the implication of the beta3-adrenoceptor (beta3-AR) pathway in human septic myocardium and a murine model of sepsis, a condition associated with myocardial depression. METHODS AND RESULTS: beta3-AR and eNOS protein abundance were increased (332+/-66.4% and 218+/-39.3; P<0.05) in hearts from septic patients. The effect of BRL37344, a beta3-AR-preferential agonist, was analyzed by videomicroscopy on the contractility of neonatal mouse ventricular myocytes (NMVM) incubated with conditioned medium from LPS-stimulated cultured macrophages (Mc-LPS+ medium). Stimulation of untreated NMVM with BRL37344 dose-dependently decreased the amplitude of contractile shortening (P<0.05). This response was abolished by L-NAME (NOS inhibitor). Incubation in Mc-LPS+ medium potentiated the depressing effect of BRL37344 (P<0.05) as well as of SR58611A (P<0.05) in wild-type myocytes. Importantly, the contractile depression was abrogated in cardiomyocytes from beta3-AR KO mice. CONCLUSIONS: beta3-AR are upregulated during sepsis in the human myocardium and by cytokines in murine cardiomyocytes, where they mediate an increased negative inotropic response to beta3 agonists. Activation of the beta3-AR pathway by catecholamines may contribute to the myocardial dysfunction in sepsis.     

Altered beta-adrenergic receptor gene regulation and signaling in chronic heart failure

J. D. Port and M. R. Bristow. Altered Beta-adrenergic Receptor Gene Regulation and Signaling in Chronic Heart Failure. Journal of Molecular and Cellular Cardiology (2001) 33, 887-905. Beta adrenergic receptors (beta -ARs) are critical regulators of cardiac function in both normal and pathophysiological states. Under normal conditions, beta -ARs and their signaling pathways modulate both the rate and force of myocardial contraction and relaxation, allowing individuals to respond appropriately to physiological stress or exercise. However, in chronic heart failure, sustained activation of the beta -AR signaling pathways can have overtly negative biological consequences. This notion is reinforced by the positive outcomes of a number of clinical trials demonstrating the usefulness of beta-blocker therapy in chronic congestive heart failure. During the last few years, significant progress has been made in understanding the molecular biological basis of beta -AR function, both at the biochemical and genetic levels. In this review, the biological basis of adrenergic signaling and how this changes in heart failure is discussed. Aspects of adrenergic receptor pharmacology relevant to heart failure are reviewed, including the recently emerging differences described for beta(1)- v beta(2)-AR signaling pathways. Highlighting these differences is recent evidence that over-stimulation of the beta(1)-AR pathway in cardiac myocytes appears to be pro-apoptotic, whereas stimulation of the beta(2)-AR pathway may be anti-apoptotic. Overview of beta -AR gene regulation, transgenic models of beta -AR overexpression, and beta -AR polymorphisms as they relate to heart failure progression are also discussed.