Beta1- and beta2-adrenoceptor polymorphisms and cardiovascular diseases

Beta(1)- and beta(2)-adrenoceptors (AR) play a pivotal role in regulation of the cardiovascular system. Both beta-AR subtypes are polymorphic. There are two major single nucleotide polymorphisms (SNPs) in the beta(1)-AR gene: the Ser49Gly and Arg389Gly beta(1)-AR polymorphisms. In vitro, in recombinant cell systems Gly49 beta(1)-AR is much more susceptible to agonist-promoted downregulation than Ser49 beta(1)-AR, while Arg389 beta(1)-AR is three to four times more responsive to agonist-evoked stimulation than Gly389 beta(1)-AR. There are three major SNPs in the beta(2)-AR gene: the Arg16Gly, Gln27Glu and Thr164Ile beta(2)-AR polymorphisms (occur in humans only in the heterozygous form). In recombinant cell systems Gly16 beta(2)-AR is much more susceptible to agonist-promoted downregulation while Glu27 beta(2)-AR is rather resistant to agonist-induced downregulation but only in combination with Arg16, that occurs naturally extremely rare. Thr164 beta(2)-AR is three to four times more responsive to agonist-evoked stimulation than Ile164 beta(2)-AR. This review summarizes results from various studies on the possible relationship of these polymorphisms to cardiovascular diseases. At present it appears to be clear that, for cardiovascular diseases such as hypertension, coronary artery disease and chronic heart failure, beta(1)- and beta(2)-AR polymorphisms do not play a role as disease-causing genes; however, they might affect drug responses. Thus, it might be possible, by assessing the beta(1)-AR genotype, to predict responsiveness to beta(1)-AR agonist and -blocker treatment: patients homozygous for the Arg389 beta(1)-AR polymorphism should be good responders while patients homozygous for the Gly389 beta(1)-AR polymorphism should be poor responders or non-responders. Furthermore, subjects heterozygous for the Thr164Ile beta(2)-AR polymorphism exhibit blunted responses to beta(2)-AR stimulation. Finally, the Arg16Gln27 beta(2)-AR haplotype appears to be - at least in human vascular and bronchial smooth muscles - rather susceptible to agonist-induced desensitization (in contrast to the recombinant cell system findings), and might have some predictive value for poor outcome of heart failure. However, future large prospective studies have to replicate these findings in order to substantiate their clinical relevance.     

Role of the beta(3)-adrenoceptor in urine storage in the rat: comparison between the selective beta(3)-adrenoceptor agonist, CL316, 243, and various smooth muscle relaxants

The objective of this study was to compare the effects of a beta(3)-adrenoceptor (beta(3)-AR) agonist on bladder function and cardiovascular parameters in rats with those of several drugs that act on smooth muscle. CL316,243 (beta(3)-AR agonist), isoproterenol (nonselective beta-AR agonist), procaterol (beta(2)-AR agonist), verapamil (Ca(2+) antagonist), and papaverine (antispastic drug) each evoked a concentration-dependent relaxation of the detrusor in vitro. They also reduced bladder pressure in anesthetized rats, the beta-AR agonists apparently being more potent than the other drugs. Atropine (muscarinic antagonist) neither relaxed detrusor strips nor reduced bladder pressure. In anesthetized rats, CL316,243 and atropine each had only a slight influence on blood pressure and heart rate, but isoproterenol, procaterol, verapamil, and papaverine significantly affected cardiovascular function at the same dose range as that required to reduce bladder pressure. In cystometry experiments, CL316,243 (10 microg/kg i.v.), verapamil (1 mg/kg i.v.), and papaverine (1 mg/kg i.v.) all significantly prolonged micturition interval and increased bladder capacity, but did not change the residual urine volume after a micturition contraction. Procaterol (100 microg/kg i.v.) prolonged the micturition interval and increased both bladder capacity and residual urine volume (all significantly). Atropine (100 microg/kg i.v.) reduced micturition pressure and increased residual urine volume (both significantly). Because the human detrusor, like the rat detrusor, relaxes on beta(3)-AR stimulation, we conclude that this beta(3)-AR agonist may have potential in pollakiuria (frequent urination) as a therapeutic agent without cardiovascular side effects.     

Direct intra-cardiomuscular transfer of beta2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters

In chronic heart failure, down-regulation of beta-adrenergic receptor (beta-AR) occurs in cardiomyocytes, resulting in low catecholamine response and impaired cardiac function. To correct the irregularity in the beta-AR system, beta-AR gene was transduced in vivo into failing cardiomyocytes. The Epstein-Barr virus (EBV)-based plasmid vector carrying human beta2-AR gene was injected into the left ventricular muscle of Bio14.6 cardiomyopathic hamsters whose beta-AR is down-regulated in the cardiomyocytes. The echocardiographic examinations revealed that stroke volume (SV) and cardiac output (CO) were significantly elevated at 2 to 4 days after the beta2-AR gene transfer. Systemic loading of isoproterenol increased the cardiac parameters more significantly on day 2 to day 7, indicating that the adrenergic response was augmented by the genetic transduction. The same procedure did not affect the cardiac function of normal hamsters. Immunohistochemical examinations demonstrated human beta2-AR expression in failing cardiomyocytes transduced with the gene. RT-PCR analysis detected mRNA for the transgene in the heart but not in the liver, spleen, or kidney. The procedures may provide a feasible strategy for gene therapy of severe heart failure. Gene Therapy (2000) 7, 2087-2093.     

Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta2-adrenergic receptor is associated with reduced phospholamban protein.

To assess the effect of targeted myocardial beta-adrenergic receptor (AR) stimulation on relaxation and phospholamban regulation, we studied the physiological and biochemical alterations associated with overexpression of the human beta2-AR gene in transgenic mice. These mice have an approximately 200-fold increase in beta-AR density and a 2-fold increase in basal adenylyl cyclase activity relative to negative littermate controls. Mice were catheterized with a high fidelity micromanometer and hemodynamic recordings were obtained in vivo. Overexpression of the beta2-AR altered parameters of relaxation. At baseline, LV dP/dt(min) and the time constant of LV pressure isovolumic decay (Tau) in the transgenic mice were significantly shorter compared with controls, indicating markedly enhanced myocardial relaxation. Isoproterenol stimulation resulted in shortening of relaxation velocity in control mice but not in the transgenic mice, indicating maximal relaxation in these animals. Immunoblotting analysis revealed a selective decrease in the amount of phospholamban protein, without a significant change in the content for either sarcoplasmic reticulum Ca2+ ATPase or calsequestrin, in the transgenic hearts compared with controls. This study indicates that myocardial relaxation is both markedly enhanced and maximal in these mice and that conditions associated with chronic beta-AR stimulation can result in a selective reduction of phospholamban protein.     

Catecholamines relax detrusor through beta 2-adrenoceptors in mouse and beta 3-adrenoceptors in man

(-)-Isoproterenol [4-[1-hydroxy-2-[(1-methylethyl)amino]ethyl]-1,2-benzene diol hydrochloride] relaxes murine detrusor through beta-adrenoceptors (ARs); however, the beta-AR subtypes involved are unknown. beta(2)-ARs have been associated with caveolae, plasma-lemmal scaffolding domains that are absent in caveolin-1 (cav-1) knockout (KO) mice. Here, we studied detrusor responses in the absence and presence of beta-AR subtype-selective antagonists in wild-type (WT) and cav-1 KO mice. To inquire whether the murine detrusor model is relevant to man, beta-AR subtypes that mediate (-)-isoproterenol-evoked human detrusor relaxation were investigated. In WT mice, (-)-isoproterenol concentration-dependently relaxed the KCl (40 mM)-precontracted detrusor (-logEC(50)M = 8.04, E(max) = 62%). The effects of (-)-isoproterenol were surmountably antagonized by the beta(2)-AR-selective antagonist ICI 118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] (pK(B) = 9.28) but not affected by the beta(1)-AR-selective antagonist CGP 20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol] and beta(3)-AR-selective L-748,337 [(S)-M-[4-[2-[3-[3-[acetamidomethyl)phenoxy)-2-hydroxypropyl]-amino]-ethyl]-phenylbenzsulfonamide)], suggesting involvement of beta(2)-AR only. The cav-1 KO detrusor displayed significant contractile dysfunction. (-)-Isoproterenol was less potent and efficient in relaxing detrusor from cav-1 KO (-logEC(50)M, 7.76; E(max) = 44%), but ICI 118,551 caused similar antagonism (pK(B) = 9.15), suggesting that beta(2)-AR function persisted in cav-1 KO. The beta(3)-AR-selective antagonist L-748,337 in the presence of ICI 118,551 and CGP 20712 caused additional blockade of (-)-isoproterenol effects in cav-1 KO, consistent with a beta(3)-AR involvement during relaxation and suppression of this effect in WT. (-)-Isoproterenol relaxed human detrusor muscle precontracted with carbachol (-logEC(50)M = 6.39, E(max) = 52%). However, the effects of (-)-isoproterenol in human detrusor were not blocked by CGP 20712 or ICI 118,551 but antagonized by L-748,337 (pK(B) = 7.65). We conclude that murine detrusor relaxation occurs via beta(2)-AR, and loss of caveolae does not perturb beta(2)-AR function but unmasks an additional activation of beta(3)-AR. In contrast, detrusor relaxation in man is mediated exclusively via beta(3)-AR.     

Age-associated reductions in cardiac beta1- and beta2-adrenergic responses without changes in inhibitory G proteins or receptor kinases.

While an age-associated diminution in myocardial contractile response to beta-adrenergic receptor (beta-AR) stimulation has been widely demonstrated to occur in the context of increased levels of plasma catecholamines, some critical mechanisms that govern beta-AR signaling must still be examined in aged hearts. Specifically, the contribution of beta-AR subtypes (beta1 versus beta2) to the overall reduction in contractile response with aging is unknown. Additionally, whether G protein-coupled receptor kinases (GRKs), which mediate receptor desensitization, or adenylyl cyclase inhibitory G proteins (Gi) are increased with aging has not been examined. Both these inhibitory mechanisms are upregulated in chronic heart failure, a condition also associated with diminished beta-AR responsiveness and increased circulatory catecholamines. In this study, the contractile responses to both beta1-AR and beta2-AR stimulation were examined in rat ventricular myocytes of a broad age range (2, 8, and 24 mo). A marked age-associated depression in contractile response to both beta-AR subtype stimulation was observed. This was associated with a nonselective reduction in the density of both beta-AR subtypes and a reduction in membrane adenylyl cyclase response to both beta-AR subtype agonists, NaF or forskolin. However, the age-associated diminutions in contractile responses to either beta1-AR or beta2-AR stimulation were not rescued by inhibiting Gi with pertussis toxin treatment. Further, the abundance or activity of beta-adrenergic receptor kinase, GRK5, or Gi did not significantly change with aging. Thus, we conclude that the positive inotropic effects of both beta1- and beta2-AR stimulation are markedly decreased with aging in rat ventricular myocytes and this is accompanied by decreases in both beta-AR subtype densities and a reduction in membrane adenylate cyclase activity. Neither GRKs nor Gi proteins appear to contribute to the age-associated reduction in cardiac beta-AR responsiveness.     

Aryloxypropanolamine and catecholamine ligand interactions with the beta(1)-adrenergic receptor: evidence for interaction with distinct conformations of beta(1)-adrenergic receptors

Pharmacological responses to aryloxypropanolamines were examined in cells expressing rat or human beta(1)-adrenergic receptors (ARs) using adenylyl cyclase assays. The aryloxypropanolamines CGP 12177 and LY 362884, originally developed as beta(3)-AR agonists, were found to stimulate the beta(1)-AR. Interestingly, both CGP 12177 and LY 362884 exhibited an anomalous biphasic effect on beta(1)-AR. Low concentrations of either CGP 12177 or LY 362884 potently blocked isoproterenol-induced stimulation of beta(1)-AR, whereas higher concentrations of these compounds stimulated the beta(1)-AR. The unusual interaction of these aryloxypropanolamine ligands with the beta(1)-AR was further characterized using beta-AR antagonists. Activation of beta(1)-AR by CGP 12177 or LY 362884 was observed to be significantly more resistant to blockade by beta-AR antagonists compared with activation by catecholamines. These results suggest that catecholamines and aryloxypropanolamines interact with distinct active conformations of the beta(1)-AR: a state that is responsive to catecholamines and is blocked with high affinity by CGP 12177 and LY 362884, and a novel state that is activated by aryloxypropanolamines but is resistant to blockade by standard beta-AR antagonists. Moreover, dependence of antagonist affinity on agonist structure is unprecedented, and its implications on the use of beta-AR agonists such as CGP 12177 in receptor classification are discussed.     

Enhancement of cardiac function after adenoviral-mediated in vivo intracoronary β2-adrenergic receptor gene delivery

Exogenous gene delivery to alter the function of the heart is a potential novel therapeutic strategy for treatment of cardiovascular diseases such as heart failure (HF). Before gene therapy approaches to alter cardiac function can be realized, efficient and reproducible in vivo gene techniques must be established to efficiently transfer transgenes globally to the myocardium. We have been testing the hypothesis that genetic manipulation of the myocardial beta-adrenergic receptor (beta-AR) system, which is impaired in HF, can enhance cardiac function. We have delivered adenoviral transgenes, including the human beta2-AR (Adeno-beta2AR), to the myocardium of rabbits using an intracoronary approach. Catheter-mediated Adeno-beta2AR delivery produced diffuse multichamber myocardial expression, peaking 1 week after gene transfer. A total of 5 x 10(11) viral particles of Adeno-beta2AR reproducibly produced 5- to 10-fold beta-AR overexpression in the heart, which, at 7 and 21 days after delivery, resulted in increased in vivo hemodynamic function compared with control rabbits that received an empty adenovirus. Several physiological parameters, including dP/dtmax as a measure of contractility, were significantly enhanced basally and showed increased responsiveness to the beta-agonist isoproterenol. Our results demonstrate that global myocardial in vivo gene delivery is possible and that genetic manipulation of beta-AR density can result in enhanced cardiac performance. Thus, replacement of lost receptors seen in HF may represent novel inotropic therapy.     

Positive inotropic stimulation

Adrenergic receptors transduce signals through the G proteins to regulate cardiac function. The catecholamines, via alpha- and beta-adrenergic receptor (beta-AR) stimulation, may play a role in the development of heart failure. Norepinephrine and isoproterenol can induce cardiac myocyte apoptosis. Studies suggest that alpha-, beta1-, and beta2-adrenergic pathways differentially regulate cardiac myocyte apoptosis. The stimulation of beta1-AR leads to cyclic AMP-dependent apoptosis, whereas that of the beta2-AR elicits concurrent apoptosis and survival signals in cardiac myocytes coupled to Gs protein. Overexpression of alpha1-adrenergic receptors does not induce apoptosis in wild-type mice. In contrast, the heart failure observed in some murine models has to be related to an enhanced beta-AR kinase expression. These recent advances make it possible to understand the beneficial effects of beta-blockers in the treatment of chronic heart failure and provide novel therapeutic modalities through the stimulation of beta2-ARs or the inhibition of beta-AR kinase expression.     

GW427353 (solabegron), a novel, selective beta3-adrenergic receptor agonist, evokes bladder relaxation and increases micturition reflex threshold in the dog

Functional studies have demonstrated that adrenoceptor agonist-evoked relaxation is mediated primarily by beta3-adrenergic receptors (ARs) in human bladder. Thus, the use of selective beta3-AR agonists in the pharmacological treatment of overactive bladder is being explored. The present studies investigated the effects of a novel selective beta3-AR agonist, (R)-3'-[[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]ethyl]amino]-[1,1'-biphenyl]-3-carboxylic acid (GW427353; solabegron) on bladder function in the dog using in vitro and in vivo techniques. GW427353 stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human beta3-AR, with an EC50 value of 22 +/- 6 nM and an intrinsic activity 90% of isoproterenol. At concentrations of 10,000 nM, GW427353 produced a minimal response in cells expressing either beta1-ARs or beta2-ARs (maximum response <10% of that to isoproterenol). In dog isolated bladder strips, GW427353 evoked relaxation that was attenuated by the nonselective beta-AR antagonist bupranolol and 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol (SR59230A) (reported to have beta3-AR antagonist activity). The relaxation was unaffected by atenolol, a selective beta1-AR antagonist, or (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118551), a selective beta2-AR antagonist. GW427353 increased the volume required to evoke micturition in the anesthetized dog following acetic acid-evoked bladder irritation, without affecting the ability of the bladder to void. GW427353-evoked effects on bladder parameters in vivo were inhibited by bupranolol. The present study demonstrates that selective activation of beta3-AR with GW427353 evokes bladder relaxation and facilitates bladder storage mechanisms in the dog.     

Role of alpha-adrenergic receptors in the effect of the beta-adrenergic receptor ligands, CGP 12177, bupranolol, and SR 59230A, on the contraction of rat intrapulmonary artery

This study investigates the effect of the aryloxypropanolamines 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one (CGP 12177), bupranolol, and 3-(2-ethylphenoxy)-1[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate (SR 59230A) [commonly used as beta(3)- and/or atypical beta-adrenergic receptors (beta-AR) ligands] on the contractile function of rat intralobar pulmonary artery. Affinities of beta-AR ligands for alpha(1)-adrenergic receptors (alpha(1)-AR) were also evaluated using [(3)H]prazosin binding competition experiments performed in rat cortical membranes. In intralobar pulmonary artery, CGP 12177 did not modify the basal tone, but antagonized the contraction induced by the alpha(1)-AR agonist phenylephrine (PHE). In arteries precontracted with PHE, CGP 12177 elicited relaxation, whereas in those precontracted with prostaglandin F(2alpha) (PGF(2alpha)), it further enhanced contraction. CGP 12177 induced an increase in intracellular calcium concentration in pressurized arteries loaded with Fura PE-3 and precontracted with PGF(2alpha). In PGF(2alpha) precontracted arteries, phentolamine (an alpha-AR antagonist) and phenoxybenzamine (an irreversible alpha-AR antagonist) antagonized the contractile responses to PHE and CGP 12177. Both responses were also decreased by bupranolol and SR 59230A. Specific [(3)H]prazosin binding was displaced by CGP 12177, bupranolol, and SR 59230A with pK(i) values of 5.2, 5.7, and 6.6, respectively. In contrast, (+/-)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium (BRL 37344) and disodium 5-[(2R)-2-([(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243) (nonaryloxypropanolamines beta(3)-AR agonists) displayed very low affinity for [(3)H]prazosin binding sites (pK(i) values below 4). These data suggest that CGP 12177 exhibits partial agonist properties for alpha(1)-AR in rat pulmonary artery. They also show that bupranolol and SR 59230A exert an alpha(1)-AR antagonist effect. As a consequence, these aryloxypropanolamine compounds should be used with caution when investigating the role of beta(3)- and atypical beta-AR in the regulation of vascular tone.     

Pharmacological characterization of KUR-1246, a selective uterine relaxant

The aim of the present study was to evaluate the efficacy and beta 2-adrenoceptor (AR) selectivity of KUR-1246, a new uterine relaxant. Inhibition of spontaneous or drug-induced uterine contractions by KUR-1246 was evaluated in pregnant rats and rabbits by an organ bath method or by a balloon method. The selectivity of KUR-1246 was assessed simultaneously in organs isolated from late-pregnant rats. The affinity of KUR-1246 for human beta 1-, beta 2-, and beta 3-ARs was determined using two radioligands. KUR-1246 suppressed both spontaneous and drug-induced contractions in isolated uteri, the rank order of potency being isoproterenol > KUR-1246 > terbutaline > ritodrine. ICI-118551 (selective beta 2-AR antagonist) competitively antagonized the KUR-1246-induced inhibition of spontaneous uterine contractions, but CGP-20712A (selective beta 1-AR antagonist) and SR-58894A (selective beta 3-AR antagonist) did not. All beta-AR agonists tested produced significant inhibition of spontaneous uterine contractions in vivo: ED(30) value for KUR-1246 was 0.13 microg/kg/min, a potency about 6 times and 400 times greater than that of terbutaline and ritodrine, respectively. In contrast, the positive chronotropic effect was minimal in KUR-1246-treated rats. KUR-1246 displaced radioligand binding to beta 1-, beta 2-, and beta 3-ARs, the pK(i) values being 5.75 +/- 0.03, 7.59 +/- 0.08, and 4.75 +/- 0.03 for beta 1-, beta 2-, and beta 3-ARs, respectively. For the selectivity of KUR-1246 for human beta 2-AR, we obtained values of 39.2 ([IC(50) for beta 1-AR]/[IC(50) for beta 2-AR]) and 198.2 ([IC(50) for beta 3-AR]/[IC(50) for beta 2-AR]), indicating an apparently higher affinity for human beta 2-AR than for other beta-AR subtypes. The present study clearly demonstrated that KUR-1246 is a more selective beta 2-AR agonist than the drugs presently used for relaxing uterine muscle.     

Estrogen reduces cardiac injury and expression of beta1-adrenoceptor upon ischemic insult in the rat heart

To test the hypothesis that estrogen confers cardioprotection by suppressing the expression of beta-adrenoceptor (beta-AR), we first correlated the infarct size in response to ischemic insult and beta-AR stimulation with the expression of beta(1)-AR in sham, ovariectomized (Ovx) and estrogen replaced (Ovx + E(2)) rats. When beta-AR is being activated during ischemia, the infarct size was significantly greater in Ovx than in the sham and Ovx + E(2) rats. There is a negative correlation between the infarct size and the expression level of beta(1)-AR as revealed by Western blotting and supported by binding analysis. Incubation of ventricular myocytes from Ovx rats with estrogen at 10(-9) M for 24 and 48 h, but not 12 h, significantly reduced lactate dehydrogenase release when the myocytes are subjected to simulated ischemia. The cardioprotective effect of 24 h estrogen incubation was accompanied by a reduction in the protein expression level of beta(1)-AR, which is estrogen receptor-dependent, whereas the lack of protection of 12-h estrogen incubation was not accompanied by any alterations in the expression level of beta(1)-AR. Together, the result from present study suggested that it is most likely that the cardioprotective effect of long-term estrogen replacement is due to suppressing the enhanced expression of cardiac beta(1)-AR in the Ovx rats, which in turn reduces cardiac injury when beta-AR is activated by sympathetic hyperactivity during ischemia. Therefore, suppression of the enhanced expression of cardiac beta(1)-AR in Ovx rats represents a novel cardioprotective mechanism of estrogen replacement therapy.     

Selectivity and potency of agonists for the three subtypes of cloned human beta-adrenoceptors expressed in Chinese hamster ovary cells

The selectivities, potencies and efficacies of beta3-adrenoceptor (beta3-AR) agonists on human three beta-AR subtypes expressed in Chinese hamster ovary (CHO) cells were investigated using radioligand binding assay and cyclic AMP (cAMP) accumulation assay. The three beta-AR subtypes showed the nature of G protein-coupled receptors with the constitutive activity. BRL37344, CL-316,243 and a newly synthesized beta3-AR agonist N-5984, 6-[2-(R)-[[2-(R)-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-2,3-dihydro-1,4-benzodioxine-2-(R)-carboxylic acid, were compared for the potency and selectivity for the beta3-AR. In the radioligand binding assay, the affinity of N-5984 for beta3-ARs was 14, 70 and 220 times more potent than those of BRL37344, isoproterenol and CL-316,243, respectively. N-5984 had higher selectivity than BRL37344 for human beta3-ARs compared with either for beta1-ARs or beta2-ARs. N-5984 showed higher potency and intrinsic activity of cAMP production than BRL37344 in CHO cells expressing the beta3-ARs. CL-316,243 had almost no activity of cAMP production in CHO cells expressing any subtype of beta-ARs. These results indicate that N-5984 is the most potent and selective agonist for human beta3-ARs than any other agonists tested.     

Mechanisms of impaired beta-adrenoceptor-induced airway relaxation by interleukin-1beta in vivo in the rat.

We studied the in vivo mechanism of beta-adrenergic receptor (beta-AR) hyporesponsiveness induced by intratracheal instillation of interleukin-1beta (IL-1beta, 500 U) in Brown-Norway rats. Tracheal and bronchial smooth muscle responses were measured under isometric conditions ex vivo. Contractile responses to electrical field stimulation and to carbachol were not altered, but maximal relaxation induced by isoproterenol (10(-6)-10(-5) M) was significantly reduced 24 h after IL-1beta treatment in tracheal tissues and to a lesser extent, in the main bronchi. Radioligand binding using [125I]iodocyanopindolol revealed a 32+/-7% reduction in beta-ARs in lung tissues from IL-1beta-treated rats, without any significant changes in beta2-AR mRNA level measured by Northern blot analysis. Autoradiographic studies also showed significant reduction in beta2-AR in the airways. Isoproterenol-stimulated cyclic AMP accumulation was reduced by IL-1beta at 24 h in trachea and lung tissues. Pertussis toxin reversed this hyporesponsiveness to isoproterenol but not to forskolin in lung tissues. Western blot analysis revealed an IL-1beta-induced increase in Gi(alpha) protein expression. Thus, IL-1beta induces an attenuation of beta-AR-induced airway relaxation through mechanisms involving a reduction in beta-ARs, an increase in Gi(alpha) subunit, and a defect in adenylyl cyclase activity.     

Adenovirus-mediated gene transfer of the beta2-adrenergic receptor to donor hearts enhances cardiac function.

Gene transfer to modify donor heart function during transplantation has significant therapeutic implications. Recent studies by our laboratory in transgenic mice have shown that overexpression of beta2-adrenergic receptors (beta2-ARs) leads to significantly enhanced cardiac function. Thus, we investigated the functional consequences of adenovirus-mediated gene transfer of the human beta2-AR in a rat heterotopic heart transplant model. Donor hearts received 1 ml of solution containing 1 x 1010 p.f.u. of adenovirus encoding the beta2-AR or an empty adenovirus as a control. Five days after transplantation, basal left ventricular (LV) pressure was measured using an isolated, isovolumic heart perfusion apparatus. A subset of hearts was stimulated with the beta2-AR agonist, zinterol. Treatment with the beta2-AR virus resulted in global myocardial gene transfer with a six-fold increase in mean beta-AR density which corresponded to a significant increase in basal contractility (LV + dP/dtmax, control: 3152.1 +/- 286 versus beta2-AR, 6250.6* +/- 432.5 mmHg/s; n = 10, *P < 0.02). beta2-AR overexpressing hearts also had higher contractility after zinterol administration compared with control hearts. Our results indicate that myocardial function of the transplanted heart can be enhanced by the adenovirus-mediated delivery of beta2-ARs. Thus, genetic manipulation may offer a novel therapeutic strategy to improve donor heart function in the post- operative setting.     

Real-time optical recording of beta1-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol

Antagonists of beta-adrenergic receptors (beta-ARs) have become a main therapeutic regimen for the treatment of heart failure even though the mechanisms of their beneficial effects are still poorly understood. Here, we used fluorescent resonance energy transfer-based (FRET-based) approaches to directly monitor activation of the beta(1)-AR and downstream signaling. While the commonly used beta-AR antagonists metoprolol, bisoprolol, and carvedilol displayed varying degrees of inverse agonism on the Gly389 variant of the receptor (i.e., actively switching off the beta(1)-AR), surprisingly, only carvedilol showed very specific and marked inverse agonist effects on the more frequent Arg389 variant. These specific effects of carvedilol on the Arg389 variant of the beta(1)-AR were also seen for control of beating frequency in rat cardiac myocytes expressing the 2 receptor variants. This FRET sensor permitted direct observation of activation of the beta(1)-AR in living cells in real time. It revealed that beta(1)-AR variants dramatically differ in their responses to diverse beta blockers, with possible consequences for their clinical use.     

beta3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current

beta3-adrenergic receptor (beta3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of beta3-AR in the human atrium. Unexpectedly, beta3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (I Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I(Ca,L) and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective beta3-AR agonists, SR58611 and BRL37344, and a beta3-AR partial agonist, CGP12177, stimulated I(Ca,L) in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The beta3-AR agonists also increased contractility but with a much lower efficacy (approximately 10%) than isoprenaline. The beta3-AR antagonist L-748,337, beta1-/beta2-AR antagonist nadolol, and beta1-/beta2-/beta3-AR antagonist bupranolol were used to confirm the involvement of beta3-ARs (and not beta1-/beta2-ARs) in these effects. The beta3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I(Ca,L) stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, beta3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.