beta-AR Antagonist Research Articles

Beta3-Adrenergic stimulation produces a decrease of cardiac contractility ex vivo in mice overexpressing the human beta3-adrenergic receptor.

The regulation of cardiac function by catecholamines involves three populations of beta-adrenoceptor (beta-AR). beta(1)- and beta(2)-AR stimulations produce an increase in contractility and beta(3)-AR stimulation mediates a negative inotropic effect in human ventricular muscle. Because of the lack of suitable animal models, we have generated transgenic mice with cardiac-specific expression of the human beta(3)-AR (TG beta(3) mice). TG beta(3) mice were produced by microinjection of the human beta(3)-AR under the control of the alpha myosin heavy chain promoter. Phenotypic analyses comprised beta(3)-AR mRNA and protein determinations, histological studies, electrocardiogram, contractility and cyclic nucleotide measurements. TG beta(3) mice presented no histological evidence of myocyte hypertrophy or fibrogenesis. In basal conditions, TG beta(3) mice were characterized by an increase in heart rate and an acceleration of twitch parameters without modification of its amplitude. beta(3)-AR agonists (CL 316243, SR 58611A) decreased contractility at low concentrations (1-100 nM). At high concentrations, the negative inotropic effect was abolished. Pretreatment with nadolol, a beta(1)/beta(2)-AR blocker, blunted the rebound in peak tension elicited by beta(3)-AR agonists suggesting a non-specific action of these compounds on beta(1)- and beta(2)-AR. The involvement of beta(3)-AR in the negative inotropic effect was confirmed by the pretreatment with bupranolol, a non-selective beta-AR antagonist, which fully abolished the effects of SR 58611A. The negative inotropic effect was associated with an increase in intracellular cGMP level. We conclude that cardiac overexpression of beta(3)-AR in mice reproduces ex vivo the negative inotropic effects obtained with beta(3)-AR stimulation in human ventricular tissues.

Interleukin-6 Family of Cytokines Mediates Isoproterenol-induced Delayed STAT3 Activation in Mouse Heart

This study was aimed to determine whether beta-adrenergic receptor (beta-AR) stimulated by isoproterenol (ISO) activates signal transducers and activators of transcription (STAT) in mouse heart and, if so, to examine the underlying mechanism. We found that treatment of adult male mice by ISO (15 mg/kg body weight, intraperitoneal) caused a delayed STAT3 activation (at 60-120 min), which was fully abolished by beta-AR antagonist, propranolol. ISO-induced phosphorylation of STAT3 was markedly enhanced by phosphodiesterase inhibitor amrinone, indicating that cAMP is critically involved in beta-AR-mediated STAT3 activation. In addition, beta-AR stimulation significantly increased gene expression of interleukin-6 (IL-6) family of cytokines (IL-6, leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin-1). IL-6 protein levels in serum and mouse myocardium were also significantly increased in response to ISO treatment. In cultured cardiac fibroblasts, IL-6 level was enhanced significantly after ISO (10-6 mol/liter) stimulation for 2 h and then peaked at 12 h, whereas the response of IL-6 in cultured cardiomyocytes to ISO stimulation was not significant, suggesting that ISO-induced increase in IL-6 is primarily from cardiac fibroblasts rather than cardiomyocytes. Most importantly, IL-6 could activate STAT3 in a time-dependent manner in cultured cardiomyocytes, and inhibition of IL-6 level by anti-IL-6-neutralizing antibody clearly attenuated ISO-induced phosphorylation of STAT3 in myocardium. Taken together, these results indicate that beta-AR stimulation leads to a delayed STAT3 activation via an IL-6 family of cytokine-mediated pathway and that cardiac fibroblasts, but not cardiomyocytes, is probably the predominant source of IL-6 in response to ISO stimulation in mouse myocardium.

Evaluation of beta3-adrenoceptor-mediated relaxation in intact and endotoxin-treated equine digital veins.

To investigate the functional expression of beta3-adrenoceptors (beta3-ARs) in equine digital veins (EDVs) and to examine whether beta3-AR relaxation was altered in EDVs incubated with endotoxin. Forelimbs obtained from 30 horses. Forelimbs were obtained from horses in an abattoir. Equine digital veins were carefully removed from distal portions of the forelimbs. Rings of dissected EDVs were mounted in 5-mL organ baths to record isometric tension in the presence of various beta3-AR agonists (SR 58611A, ZD 2079, and ZM 215001). In intact EDVs, isoprenaline, SR 58611A, ZD 2079, and ZM 215001 induced concentration-dependent relaxation. Isoprenaline and SR 58611A-induced relaxations were reduced or unaffected by nadolol, respectively. In intact EDVs, SR 58611A-induced relaxation was significantly reduced in the presence of 2 microM ZM 215001 (used as a beta3-AR antagonist). In endothelium-denuded EDVs or intact EDVs in the presence of a nitric oxide synthase inhibitor, isoprenaline and SR 58611A-induced relaxations were significantly decreased. The endothelium-independent relaxation to SR 58611A was significantly inhibited in the presence of ZM 215001. In endotoxin-treated EDV, isoprenaline- and SR 58611A-induced relaxations were significantly reduced. In these conditions, cycloheximide (a protein synthesis inhibitor) and ibuprofen (a cyclooxygenase inhibitor) restored the relaxant response to SR 58611A. Beta3-adrenoceptors are functionally expressed in EDVs. Incubation in the presence of endotoxin, used as an in vitro model of laminitis, induced an alteration of beta-AR-mediated relaxations in EDVs, which could be the consequence of cyclooxygenase induction and subsequent prostanoid production.

Role of nitric oxide in beta3-adrenoceptor activation on basal tone of internal anal sphincter.

Effects of activation of beta3-adrenoceptor (beta3-AR) have not been determined in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The effects of disodium (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), a selective beta3-AR agonist, on the basal smooth muscle tone and direct release of nitric oxide (NO) by circular smooth muscle strips of the opossum IAS were determined. We also examined the presence of endothelial nitric oxide synthase (eNOS) protein by Western blot studies. CL 316243 produced a concentration-dependent relaxation of the smooth muscle that remained unmodified by different neurohumoral antagonists. The smooth muscle relaxation by CL 316243 was selectively antagonized by L 748337, a beta3-AR antagonist. Such relaxation was several times longer than by isoproterenol. The effect of CL 316243 was significantly attenuated by a nonselective NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) and by putative inhibitor of eNOS l-N5-(1-iminoethyl)-ornithine dihydrochloride (l-NIO). Inhibitors of iNOS [N-(3-aminomethyl)benzyl acetamide 2HCl] and nNOS [1-[2-(trifluoromethylphenyl)imidazole]] had no effect on this relaxation. Relaxation of the IAS smooth muscle induced by CL 316243 was accompanied by an increased release of NO; this was attenuated by l-NNA and l-NIO. In addition, Western blot studies revealed the presence of eNOS in the circular smooth muscle of the IAS. These data demonstrate potent and protracted IAS smooth muscle relaxation by beta3-AR activation, which is partly transduced via NOS, possibly smooth muscle eNOS. Multiple signal-transduction pathways including NOS activation may explain the characteristic IAS relaxation by beta3-AR activation. The studies may have therapeutic implications in anorectal motility disorders.

Cardiac-specific overexpression of human beta2 adrenoceptors in mice exposes coupling to both Gs and Gi proteins.

1. Left atrial strips from transgenic (TG4) mice with cardiac-specific overexpression ( approximately 200-fold) of the beta(2) adrenoceptor (beta(2)AR) were isolated, and their isometric force of contraction (F(c)) in response to electrical stimulation was measured. 2. The betaAR agonist isoprenaline elicited negative inotropic responses in all left atrial strips; in 6/11 preparations, it also had a small positive inotropic effect. This 'up-phase' was observed from 0.1 to 10 nM, with the 'down-phase' occurring at higher concentrations. Both phases were mediated by beta(2)AR, as shown by their sensitivity to the beta(2)AR antagonist ICI-118,551 (100 nM; pA(2) 8.60+/-0.07, 8.45+/-0.19, for 'up-phase' and 'down-phase,' respectively), but not the beta(1)AR antagonist CGP-20712A (100 nM). Conversely, nontransgenic littermate preparations responded to isoprenaline treatment solely by an increase in F(c), which was beta(1)AR-mediated. 3. Pretreatment of left atrial strips with either 10 nM isoprenaline or 1 mM 8-bromo-cAMP significantly attenuated the TG4 'up-phase', while having no effect on either the TG4 'down-phase' or the littermate controls' responses. B. pertussis toxin treatment of the animals prevented isoprenaline's negative inotropic effects in TG4 preparations, but had no effect in littermate controls. 4. The findings imply that the responses of TG4 left atrium to isoprenaline are because of beta(2)AR coupling to G(s) and G(i) proteins, consistent with the model of Daaka et al., in which protein kinase A phosphorylation of the beta(2)AR causes a switch from G(s) to G(i) protein coupling.

Characterization of adrenoceptor involvement in skeletal and cardiac myotoxicity Induced by sympathomimetic agents: toward a new bioassay for beta-blockers.

Excessive levels of catecholamines have long been known to be cardiotoxic, but less well known are their toxic effects on skeletal muscle. By using an antimyosin monoclonal antibody and quantitative methods to measure the extent of myocyte necrosis, and by employing modulators of adrenoceptors (ARs), including clenbuterol, bupranolol, propranolol, bisoprolol, atenolol, ICI-118551, phenoxybenzamine, prazosin, and yohimbine, the involvement of ARs in isoproterenol-induced myotoxicity was characterized. In the myocardium, the toxic effects were predominantly mediated via the beta(1)-ARs. In the soleus muscle, it was almost solely via the beta(2)-ARs. Myotoxicity was also observed in the myocardium when challenged with the beta(2)-AR agonist clenbuterol. This was found to be mediated via sympathetic presynaptic beta(2)-ARs, leading to enhanced release of norepinephrine. This effect was abolished by prior treatment with reserpine. The skeletal muscle was found to be more sensitive to the myotoxic effects than cardiac muscle at lower doses of beta-AR agonists. These experiments introduce a new way of assaying beta-AR antagonists by classifying them according to their ability to prevent catecholamine-induced myotoxicity. Further research along these lines may deepen understanding of which beta-blockers work best in heart failure therapy.

Beta-adrenergic receptor subtypes that mediate ractopamine stimulation of lipolysis.

Ractopamine HCl is an beta-adrenergic receptor (betaAR) ligand that was recently approved for use in swine to enhance carcass leanness. The RR stereoisomer of ractopamine is the most active of the four stereoisomers exhibiting the highest affinity and signaling response. The RR isomer exhibits selective activation of the porcine beta2AR, which might limit the lipolytic response to ractopamine because the betaAR is the predominant subtype in swine adipocytes and may mediate most of the lipolytic response. Therefore, we determined the betaAR subtypes that mediate the lipolytic response to ractopamine in swine adipocytes. In order to confirm the predominant role of the beta1AR in porcine adipocytes, isoproterenol-stimulated lipolysis was inhibited by increasing doses of subtype-selective antagonists. Inhibition curves were biphasic using beta1AR antagonists (CGP 20712A and bisoprolol) and curve analysis indicated that both beta1AR an beta2AR contributed to lipolysis with 50 to 60% of the response coming from the beta1AR. Inhibition with the beta2AR antagonist clenbuterol revealed only one class of betaAR that closely approximated the kinetics of the beta1AR. When the RR isomer of ractopamine was the lipolytic agent, similar results to isoproterenol were observed, except that the estimated contribution of the beta1AR was 38%. That beta2AR antagonists did not detect a contribution of the beta2AR to lipolysis may indicate that the beta1AR masked the response to the beta2AR. Dose titration with the RR isomer in the presence of a saturating concentration of beta1AR or beta2AR antagonists indicated that each subtype was present in sufficient quantities to stimulate lipolysis near maximally. Data indicate that both the beta1AR and beta2AR are functionally linked to lipolysis in swine adipocytes and that ractopamine activates each subtype. The RR isomer of ractopamine stimulated adenosine 3',5'-cyclic phosphate accumulation with equal efficacy to isoproterenol through the cloned porcine beta2AR, but was only 35% as efficacious through the cloned porcine beta1AR. These data confirm the beta2AR selectivity of the RR stereoisomer, but suggest the partial agonism through the beta1AR is sufficient to activate lipolysis through both subtypes in swine adipocytes.

Chronic beta2-adrenergic receptor stimulation increases proliferation of human cardiac fibroblasts via an autocrine mechanism.

The aim of this study was to determine whether chronic beta-adrenergic receptor (beta-AR) stimulation induces proliferation of human cardiac fibroblasts and to investigate the mechanism(s) involved. In vitro cultures of human cardiac fibroblasts were established from biopsies of right atrial appendage. RT-PCR analysis and pharmacological studies demonstrated that these cells express predominantly the beta(2)-AR subtype coupled to activation of adenylyl cyclase and p44/42 mitogen-activated protein kinase (MAPK). Proliferation was determined by cell counting over a 6-day period in medium containing 2.5% fetal calf serum (control) or supplemented with the non-selective beta-AR agonist isoproterenol (ISO). ISO induced a concentration-dependent increase in cardiac fibroblast proliferation, which was maximal at 1 micromol/l. This increased proliferation was inhibited by the beta(2)-AR-selective antagonist ICI-118,551, but not the beta(1)-AR-selective antagonist atenolol. Direct activation of adenylyl cyclase alone (0.1-10 micromol/l forskolin) stimulated cyclic AMP production and MAPK activation, but did not induce cell proliferation. Since catecholamines are not considered to be 'classical' growth factors, we subsequently investigated whether beta(2)-AR stimulation results in secretion of growth factors that are able to stimulate proliferation in an autocrine manner. Conditioned medium obtained from cardiac fibroblasts treated with ISO for 48 h increased proliferation of parallel cultures of fibroblasts in the presence of the beta-AR antagonist alprenolol. Heat-treatment of this conditioned medium fully prevented the increase in cell proliferation, indicating that the autocrine factor(s) are heat-sensitive proteins. Chronic beta(2)-AR stimulation increases proliferation of human cardiac fibroblasts via a mechanism involving increased secretion of heat-sensitive growth factors.

Desensitization of alpha 2A-adrenoceptor signalling by modest levels of adrenaline is facilitated by beta 2-adrenoceptor-dependent GRK3 up-regulation.

(1) Adrenaline (ADR) and noradrenaline (NA) can simultaneously activate inhibitory alpha(2)- and stimulatory beta-adrenoceptors (AR). However, ADR and NA differ significantly in that ADR is a potent beta(2)-AR agonist while NA is not. Only recently has the interaction resulting from the simultaneous activation of alpha(2)- and beta(2)-AR been examined at the cellular level to determine the mechanisms of alpha(2)-AR regulation following concomitant activation of both alpha(2)- and beta(2)-ARs by chronic ADR. (2) This study evaluates beta(2)-AR regulation of alpha(2A)-AR signalling following chronic ADR (300 nM) and NA (1 and 30 micro M) treatments of BE(2)-C human neuroblastoma cells that natively express both beta(2)- and alpha(2A)-ARs. (3) Chronic (24 h) treatment with ADR (300 nM) desensitized the response to the alpha(2A)-AR agonist, brimonidine, in BE(2)-C cells. Addition of the beta-AR antagonist, propranolol, blocked the ADR-induced alpha(2A)-AR desensitization. Unlike ADR, chronic NA (1 micro M) treatment had no effect on the alpha(2A)-AR response. However if NA was increased to 30 micro M for 24 h, alpha(2A)-AR desensitization was observed; this desensitization was partially reversed by propranolol. (4) Chronic ADR (300 nM) treatment reduced alpha(2A)-AR binding levels, contributing to the alpha(2A)-AR desensitization. This decrease was prevented by addition of propranolol during ADR treatment. Chronic NA (30 micro M), like ADR, treatment lowered specific binding, whereas 1 micro M NA treatment was without effect. (5) Chronic ADR treatment produced a significant increase in GRK3 levels and this was blocked by propranolol or GRK2/3 antisense DNA treatment. This antisense DNA, common to both GRK2 and GRK3, also blocked chronic ADR-induced alpha(2A)-AR desensitization and down-regulation. (6) Acute (1 h) ADR (300 nM) or NA treatment (1 micro M) produced alpha(2A)-AR desensitization. The desensitization produced by acute treatment was beta-AR independent, as it was not blocked by propranolol. (7) We conclude that chronic treatment with modest levels of ADR produces alpha(2A)-AR desensitization by mechanisms that involve up-regulation of GRK3 and down-regulation of alpha(2A)-AR levels through interactions with the beta(2)-AR.

Functional and molecular characterization of beta-adrenoceptors in the internal anal sphincter.

The purpose of the present study was to characterize different beta-adrenoceptors (beta-ARs) and determine their role in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The beta-AR subtypes in the opossum IAS were investigated by functional in vitro, radioligand binding, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR) studies. ZD 7114 [(S)-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-(2-methoxyethyl)phenoxyacetamide], a selective beta(3)-AR agonist, caused a potent and concentration-dependent relaxation of the IAS smooth muscle that was antagonized by the beta(3)-AR antagonist SR 59230A [1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride]. Conversely, the IAS smooth muscle relaxation caused by beta(1)- and beta(2)-AR agonists (xamoterol and procaterol, respectively) was selectively antagonized by their respective antagonists CGP 20712 [(+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt] and ICI 118551. Saturation binding of [(125)I]iodocyanopindolol to beta-AR subtypes revealed the presence of a high-affinity site (K(d1) = 96.4 +/- 8.7 pM; B(max1) = 12.5 +/- 0.6 fmol/mg protein) and a low-affinity site (K(d2) = 1.96 +/- 1.7 nM; B(max2) = 58.7 +/- 4.3 fmol/mg protein). Competition binding with selective beta-AR antagonists revealed that the high-affinity site correspond to beta(1)/beta(2)-AR and the low affinity site to beta(3)-AR. Receptor binding data suggest the predominant presence of beta(3)-AR over beta(1)/beta(2)-AR. Western blot studies identified beta(1)-, beta(2)-, and beta(3)-AR subtypes. The presence of beta(1)-, beta(2)-, and beta(3)-ARs was further demonstrated by mRNA analysis using RT-PCR. The studies demonstrate a comprehensive functional and molecular characterization of beta(1)-, beta(2)-, and beta(3)-ARs in IAS smooth muscle. These studies may have important implications in anorectal and other gastrointestinal motility disorders.

Esmolol improves left ventricular function via enhanced beta-adrenergic receptor signaling in a canine model of coronary revascularization.

Recent American Heart Association guidelines highlight the paucity of data on effectiveness and/or mechanisms underlying use of beta-adrenergic receptor (beta AR) antagonists after acute coronary syndromes in patients subsequently undergoing revascularization. It is important to assess whether beta AR antagonists might protect the heart and improve ventricular function in this scenario. The authors therefore used esmolol (an ultra-short-acting beta AR antagonist) to determine whether beta AR antagonist treatment improves left ventricular function in a canine model of acute reversible coronary ischemia followed by coronary reperfusion during cardiopulmonary bypass (CPB). The authors also tested whether the mechanism includes preserved beta AR signaling. Dogs were randomized to either esmolol or saline infusions administered during CPB (n = 29). Pre-CPB and end-CPB transmyocardial left ventricular biopsies were obtained; plasma catecholamine concentrations, myocardial beta AR density, and adenylyl cyclase activity were measured. In addition, left ventricular systolic shortening and postsystolic shortening were determined immediately prior to each biopsy. While beta AR density remained unchanged in each group, isoproterenol-stimulated adenylyl cyclase activity decreased 26 +/- 6% in the control group but increased 38 +/- 10% in the esmolol group (pre-CPB to end-CPB, mean +/- SD, P = 0.0001). Left ventricular systolic shortening improved in both groups after release of coronary (LAD) ligature; however, the esmolol group increased to 72 +/- 23% of pre-CPB values compared to 48 +/- 12% for the control group (P = 0.0008). These data provide prospective evidence that esmolol administration results in improved myocardial function. Furthermore, the mechanism appears to involve enhanced myocardial beta AR signaling.

Norepinephrine Content And Adrenoceptor Function In The Bladder Of Cats With Feline Interstitial Cystitis

Interstitial cystitis is a chronic syndrome affecting humans and domestic animals, including cats (feline interstitial cystitis). The aggravation of interstitial cystitis symptoms by stress suggests involvement of the sympathetic nervous system. Studies have identified increased sympathetic nervous system activity in patients with interstitial cystitis but to our knowledge effects on bladder function have not been reported. To address this question we measured bladder norepinephrine (NE) content, the electrical field stimulation flux of NE and acetylcholine (ACh), and the effects of feline interstitial cystitis on adrenoceptor (AR) mediated bladder strip contractility. Bladders were obtained from healthy cats and cats with feline interstitial cystitis. In experiment 1 bladder tissue NE content was determined and the simultaneous release of 3H-NE and 14C-ACh in perfusion bath effluent after electrical field stimulation was measured. NE and ACh release was calculated from the area under the efflux curve. In experiment 2 electrical field stimulation induced contractility of bladder body strips was measured in the presence of 100 nM. to 25 microM. NE only or combined with atipamezole (an alpha2-AR antagonist), propranolol (a beta-AR antagonist) or phentolamine (an alpha-AR antagonist). Antagonists were added to the bath at least 15 minutes before stimulation, after which NE was added in cumulative doses and dose response curves were constructed. Significant increases in NE content and efflux in the absence of alterations in ACh efflux were identified. In the bladder strip studies decreased alpha2 and beta1-AR function was found in strips from cats with feline interstitial cystitis, whereas beta3 or atypical beta-ARs were tentatively identified. These results support and extend previous studies by identifying an effect of increased sympathetic activity on bladder function in cats with feline interstitial cystitis.

Tyr199 in transmembrane domain 5 of the β2-adrenergic receptor interacts directly with the pharmacophore of a unique fluorenone-based antagonist

Mutagenesis of the beta2-adrenergic receptor (beta2AR) has suggested that amino acids in transmembrane domain 5 (TMD 5) play an important role in the interaction of the receptor with the catechol end of adrenergic agonists. However, little direct biochemical evidence for the interaction of any beta2AR agonist or antagonist with TMD 5 has been reported. To identify receptor amino acids that contribute to the beta2AR antagonist binding site, we identified the precise amino acid photoinsertion site of a novel carazolol-like fluorenone antagonist photoaffinity label, [125I]iodoaminoflisopolol ([125I]IAmF). A unique property of this photolabel is that the photoreactive centre is also the binding pharmacophore, which corresponds to the catechol end of related beta2AR agonists. [125I]IAmF specifically photolabels membrane-bound and purified beta2AR from a baculovirus/Spodoptera frugiperda (fall armyworm) ('Sf9') expression system. When the photolabelled beta2AR was cleaved by trypsin or Factor Xa, 30 kDa labelled peptides were generated. On the basis of concanavalin A binding and amino acid sequencing, these contain the N-terminus of the beta2AR, including TMDs 1-5. Further cleavage of the 30 kDa peptides with endoproteinase Lys-C generated a 4 kDa labelled peptide with an N-terminal amino acid sequence between TMDs 4 and 5. Radiosequencing of this peptide demonstrated that the precise [125I]IAmF photoinsertion site was Tyr(199) in TMD 5. Since the photoreactive centre and the binding pharmacophore of IAmF are the same, these data demonstrate that Tyr(199) interacts with the planar fluorenone moiety of a carazolol-like beta2AR antagonist, and contributes significant new information regarding the binding site for beta2AR antagonists.

Beta(1)-Adrenoceptors compensate for beta(3)-adrenoceptors in ileum from beta(3)-adrenoceptor knock-out mice.

1. This study examines beta(1)-, beta(2)- and beta(3)-adrenoceptor (AR)-mediated responses, mRNA levels and radioligand binding in ileum from beta(3)-AR knock-out (-/-) (KO) and wild type (+/+) (FVB) mice. 2. In KO and FVB mice, SR59230A (100 nM) (beta(3)-AR antagonist) antagonized responses to (-)-isoprenaline in both KO and FVB mice. (-)-Isoprenaline mediated relaxation of ileum was antagonized weakly by ICI118551 (100 nM) (beta(2)-AR antagonist). Responses to (-)-isoprenaline were more strongly antagonized by CGP20712A (100 nM) (beta(1)-AR antagonist), propranolol (1 microM) (beta(1)-/beta(2)-AR antagonist), carvedilol (100 nM) (non-specific beta-AR antagonist), and CGP12177A (100 nM) (beta(1)-/beta(2)-AR antagonist) in ileum from KO than in FVB mice. 3. Responses to CL316243 (beta(3)-AR agonist) in ileum from FVB mice were antagonized by SR59230A (100 nM) but not by propranolol (1 microM) or carvedilol (100 nM). CL316243 was ineffective in relaxing ileum from KO mice. 4. CGP12177A had no agonist actions in ileum from either KO or FVB mice. 5. beta(1)-AR mRNA levels were increased 3 fold in ileum from KO compared to FVB mice. This was associated with an increased maximum number of beta(1)-/beta(2)-AR binding sites (B(max)). beta(2)-AR mRNA levels were unaffected while no beta(3)-AR mRNA was detected in KO mice. 6. In mouse ileum, beta(3)-ARs and to a lesser extent beta(1)-ARs are the predominant adrenoceptor subtypes mediating relaxation in ileum from FVB mice. In KO mice beta(1)-ARs functionally compensate for the lack of beta(3)-ARs, and this is associated with increased beta(1)-AR mRNA and levels of binding.

Thiazolidinediones inhibit the expression of beta3-adrenergic receptors at a transcriptional level.

The effect of the thiazolidinediones (TZDs) darglitazone and troglitazone on beta3-adrenergic receptor (AR) expression was studied in cultured cell lines representing several tissues. After 24 h of exposing HIB-1B brown adipocytes to 30 micromol/l darglitazone or 20 micromol/l troglitazone, beta3-AR mRNA levels were reduced by 75%. This effect was significant within 1 h of exposure to a maximal dose of these drugs, with the full effect obtained within 10 h. The darglitazone ID50 was approximately 10 nmol/l, similar to the Kd of TZDs binding to peroxisome proliferator-activated receptor-gamma (PPAR-gamma). These drugs also decreased beta3-AR mRNA in 3T3-F442A white adipocytes, but not in SK-N-MC cells, which lack PPAR-gamma2. A luciferase reporter gene containing 1.4 kb of 5' flanking sequence of the mouse beta3-AR was transiently transfected, with or without PPAR-gamma2, in SK-N-MC cells. The vigorous expression of luciferase driven by the beta3-AR gene sequence was inhibited by TZDs in a PPAR-gamma2-dependent manner. The half-lives of gamma3-AR precursor RNA and mRNA were short, approximately 40 and approximately 100 min, respectively, and remained unaffected by TZD treatment. Exposure of HIB-1B cells to 30 micromol/l darglitazone was associated with reduced beta3-AR mRNA levels, as well as decreased response of uncoupling protein 1 to norepinephrine + propranolol (a beta1 beta2-AR antagonist) or the specific beta3-AR agonist CL 316, 243. Both the beta3-AR mRNA level and response to these stimuli fully recovered by 24 h of removing the drug, indicating that the beta3-AR protein and its coupling to adenylyl cyclase rapidly followed the changes in mRNA. Thus, TZDs can rapidly reduce beta3-AR expression at the transcriptional level, acting through PPAR-gamma2. The rapid turnover and responses of beta3-AR to perturbations, along with numerous other factors reported to regulate its expression, suggest a tight control of beta3-AR and function. Lastly, leptin being the only other known gene suppressed by TZDs, the present studies support a concerted lipogenic effect of these drugs.

EXISTENCE OF A β3-ADRENOCEPTOR AND ITS FUNCTIONAL ROLE IN THE HUMAN URETER

We tried to determine the beta-adrenoceptor (AR) subtypes distributed in the human ureter and to clarify their functional role in ureteral relaxation. 1) Effects of beta-AR agonists on either spontaneous or KCl-induced contractions of the human ureter and the antagonism by beta-AR antagonists on isoprenaline (a non-selective beta-AR agonist)-induced effects were evaluated in vitro. 2) Displacement by beta-AR antagonists of [3H]-dihydroalprenolol binding to a membrane preparation derived from human ureteral smooth muscle was evaluated. 3) A reverse transcription polymerase chain reaction assay was performed to determine the expression of the mRNA for beta1-, beta2- and beta3-ARs in human ureteral smooth muscle. 1) Isoprenaline and procaterol (a beta2-AR agonist) concentration-dependently suppressed both spontaneous and KCl-induced contractions of the human ureter. The beta3-AR agonists, CGP-12177A and CL-316243, also suppressed these ureteral contractions, but dobutamine (a beta1-AR agonist) had little relaxing effect. The rank order of relaxing potency for the catecholamines was isoprenaline > adrenaline > noradrenaline. ICI-118,551 (a beta2-AR antagonist) only partially antagonized the isoprenaline-induced relaxation. 2) Propranolol (a non-selective beta-AR antagonist) and ICI-118,551 concentration-dependently displaced [3H]-dihydroalprenolol binding to the membrane with Ki values of 1.5 x 10-9 M and 6.3 x 10-9 M, respectively, while metoprolol (a beta1-AR antagonist) was less effective in this assay. 3) beta1-, beta2- and beta3-AR mRNAs were all expressed in human ureteral smooth muscle. The present results provide the first evidence that the beta3-AR subtype is distributed in human ureteral smooth muscle and that it, and beta2-AR, mediate the ureteral relaxation induced by adrenergic stimulation.

A High-Affinity Fluorenone-Based β2-Adrenergic Receptor Antagonist with a Photoactivatable Pharmacophore

To develop molecules capable of directly probing the catechol binding region of the beta(2)-adrenergic receptor (beta(2)AR), novel benzophenone- and fluorenone-based beta(2)AR antagonists were prepared as potential photoaffinity probes. While the benzophenone-containing ligands bound with relatively modest affinity, one of the fluorenone-based compounds, 4-(2-hydroxy-3-isopropylaminopropoxy)-7-amino-6-iodofluorenone+ ++ (iodoaminoflisopolol, IAmF), showed very high affinity for the beta(2)AR, inhibiting [(125)I]ICYP binding with an apparent K(i) of approximately 1 x 10(-)(9) M. In comparison to the benzophenone ligands, the fluorenone ligands have one additional carbon-carbon bond that creates a planar unsaturated ring system and leads to a large increase in receptor binding affinity. Unlike previous beta(2)AR photoaffinity ligands, an attractive and unique feature of the fluorenone derivative IAmF is that the large planar unsaturated ring (believed to correspond to the catechol end of other beta(2)AR ligands) serves as both the binding pharmacophore and the photoreaction center for this molecule. With this potential for directly probing the catechol binding region of the beta(2)AR, we synthesized and tested IAmF in carrier-free radioiodinated form ([(125)I]IAmF). When photoreduction was conducted at 350 nm for 20 min, [(125)I]IAmF was able to produce cross-linked products in both triethylamine and methanol, with a reactivity pattern similar to that found in benzophenone photochemistry. As a final test of suitability as a photoaffinity label, specific labeling of the beta(2)AR in membranes (protectable by 10 microM alprenolol) was demonstrated. [(125)I]IAmF represents a new class of beta(2)AR photoaffinity labels that can directly probe the catechol-analogous antagonist pharmacophore binding site in the beta(2)AR ligand binding pocket.

The negative inotropic effect of beta3-adrenoceptor stimulation in the beating guinea pig heart.

Although beta3-adrenoceptors (ARs) have been extensively characterized in brown and white adipocytes, their actions in the beating heart are unclear. We examined the effects of a beta3-AR agonist, BRL37344, on cardiac function and calcium transients in Langendorff-perfused guinea pig hearts by simultaneously measuring left ventricular (LV) pressure and Ca2+-dependent indo-1 fluorescence. BRL37344 induced a dose-dependent negative inotropic effect at concentrations from 10(-11) to 10(-8) M. Maximally, LV developed pressure decreased to 80+/-2%, +dP/dt to 81+/-2%. and -dP/dt to 81+/-3% of their respective control values (p < 0.01). The amplitude of the Ca2+ transient also decreased (to 92+/-3% of the control level; p < 0.01). The BRL37344 dose-response curve was not altered by nadolol (10(-5) M), a potent beta1- and beta2-AR antagonist, but completely suppressed by bupranolol (10(-6) M), a potent beta1-, beta2- and beta3-AR antagonist. To assess the potential role of a nitric oxide synthase (NOS) pathway, we determined whether the NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), modified the contractile response to BRL37344. L-NAME (10(-7) and 10(-4) M) attenuated the negative inotropic effects on LV developed pressure by 35 and 50%, suggesting that beta3-AR stimulation induces a negative inotropic effect on guinea pig hearts partly through a decrease in the Ca2+ transient and partly by the NOS pathway.

Ligand-independent negative chronotropic responses of rat and mouse right atria to β-adrenoceptor antagonists

Negative chronotropic effects of beta-adrenoceptor (betaAR) antagonists on right atria from reserpine-treated rats and mice were determined as a test of their inverse agonist activities. BetaAR antagonist ICI-118,551 and nonselective betaAR antagonists alprenolol, propranolol, and timolol produced negative chronotropic effects. In contrast, nonselective betaAR antagonists pindolol and nadolol as well as beta1AR-selective antagonists atenolol, acebutolol, and metoprolol did not cause a significant decrease in atrial rates. The neutral antagonist pindolol but not the inverse agonist alprenolol inhibited the negative chronotropic activities of ICI-118,551. Isoprenaline, salbutamol, and noradrenaline produced positive chronotropic effects; the chronotropic effects of isoprenaline and salbutamol but not of noradrenaline were antagonized by ICI-118,551. It is concluded that both beta1AR and beta2AR mediate positive chronotropic effects of catecholamines on rat and mouse atria but only beta2AR are constitutively active.

Expression of human (beta)3-adrenergic receptor induces adipocyte-like features in CHO/K1 fibroblasts.

It is reported here that CHO/K1 cells stably transfected with the human (beta)3 AR gene (CHO/K1-(beta)3), grown in the presence of differentiation-stimulating agents accumulate triglycerides. This lipid formation is mediated through the (beta)3 AR, since non-transfected CHO/K1 cells, or cells expressing the human (beta)2 AR, accumulate no significant amount of lipids when grown in supplemented medium. Moreover, lipid production can be inhibited significantly by the (beta) AR antagonist bupranolol. CHO/K1 cells expressing the W64R polymorphism (Trp to Arg polymorphism at position 64 of the human (beta)3 AR), which has been associated with morbid obesity, show increased lipid accumulation as compared to CHO/K1 cells expressing the wild-type (beta)3 AR. Semi-quantitative RT-PCR experiments reveal that a major gene regulating adipocyte differentiation, peroxisome-proliferator-activated-receptor (gamma) (PPAR(gamma)), is expressed in CHO/K1 cells. Concomitantly with the formation of lipid droplets, the expression of PPAR(gamma) mRNA is increased in CHO/K1-(beta)3 cells, but not in non-transfected CHO/K1 cells. We furthermore detected constitutive expression of another adipocyte-associated protein: hormone sensitive lipase, while leptin or uncoupling protein-1 transcripts were not expressed. These data suggest that the frequently used CHO/K1 fibroblasts display several preadipocyte-like features, and that the sole expression of the (beta)3 AR modifies the expression of PPAR(gamma) mRNA in these cells, and induces lipid formation under certain culture conditions.