beta-AR Agonist Research Articles

Overexpression of β2-Adrenergic Receptors cAMP-dependent Protein Kinase Phosphorylates and Modulates Slow Delayed Rectifier Potassium Channels Expressed in Murine Heart: EVIDENCE FOR RECEPTOR/CHANNEL CO-LOCALIZATION

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

Learning new tricks from old dogs: beta-adrenergic receptors teach new lessons on firing up adipose tissue metabolism.

The three beta AR (beta-adrenergic receptor) subtypes (beta(1)AR, beta(2)AR, and beta(3)AR) are members of the large family of G protein-coupled receptors, each of which is coupled to G alpha s and increases in intracellular cAMP levels. In white adipose tissues, catecholamine activation of the beta ARs leads to the mobilization of stored fatty acids and regulates release of several adipokines, whereas in brown adipose tissue they stimulate the specialized process of adaptive nonshivering thermogenesis. Noteworthy, in most models of obesity the beta AR system is dysfunctional, and its ability to stimulate lipolysis and thermogenesis are both impaired. Nevertheless, selective agonists for the beta(3)AR, a subtype that is found predominantly in adipocytes, have been able to prevent or reverse obesity and accompanying insulin resistance in animal models. Whether this is a viable therapeutic option for human obesity is much debated with regard to the existence of brown adipocytes in humans or their ability to be recruited. Nevertheless, probing the physiological changes in adrenoceptor function in rodent obesity, as well as the process by which beta(3)AR agonists promote a thermogenic shift in fuel use, have yielded unexpected new insights into beta AR signaling and adipocyte physiology. These include the recent discovery of an essential role of p38 MAPK in mediating adaptive thermogenesis, as well as the accessory role of the ERK MAPK pathway for the control of lipolysis. Because these metabolic events were traditionally ascribed solely to the cAMP/protein kinase A system, the integration of these signaling mechanisms may pose new therapeutic directions in the quest to counter the obesity epidemic in our midst.

Modeling the actions of beta-adrenergic signaling on excitation--contraction coupling processes.

Activation of the beta-adrenergic (beta-AR) signaling pathway enhances cardiac function through protein kinase A (PKA)-mediated phosphorylation of target proteins involved in the process of excitation-contraction (EC) coupling. Experimental studies of the effects of beta-AR stimulation on EC coupling have yielded complex results, including increased, decreased, or unchanged EC coupling gain. In this study, we extend a previously developed model of the canine ventricular myocyte describing local control of sarcoplasmic reticulum (SR) calcium (Ca(2+)) release to include the effects of beta-AR stimulation. Incorporation of phosphorylation-dependent effects on model membrane currents and Ca(2+)-cycling proteins yields changes of action potential (AP) and Ca(2+) transients in agreement with those measured experimentally in response to the nonspecific beta-AR agonist isoproterenol (ISO). The model reproduces experimentally observed alterations in EC coupling gain in response to beta-AR agonists and predicts the specific roles of L-type Ca(2+) channel (LCC) and SR Ca(2+) release channel phosphorylation in altering the amplitude and shape of the EC coupling gain function. The model also indicates that factors that promote mode 2 gating of LCCs, such as beta-AR stimulation or activation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), may increase the probability of occurrence of early after-depolarizations (EADs), due to the random, long-duration opening of LCC gating in mode 2.

Effects of acute and chronic administration of beta-adrenoceptor ligands on airway function in a murine model of asthma.

The clinical effects of treatment with beta-adrenoceptor (beta-AR) agonists and antagonists in heart failure vary with duration of therapy, as do the effects of beta-AR agonists in asthma. Therefore, we hypothesized that chronic effects of "beta-blockers" in asthma may differ from those observed acutely. We tested this hypothesis in an antigen (ovalbumin)-driven murine model of asthma. Airway resistance responses (Raw) to the muscarinic agonist methacholine were measured by using the forced oscillation technique. In comparison with nontreated asthmatic mice, we observed that: (i) The beta-AR antagonists nadolol or carvedilol, given as a single i.v. injection (acute treatment) 15 min before methacholine, increased methacholine-elicited peak Raw values by 33.7% and 67.7% (P < 0.05), respectively; when either drug was administered for 28 days (chronic treatment), the peak Raw values were decreased by 43% (P < 0.05) and 22.9% (P < 0.05), respectively. (ii) Chronic treatment with nadolol or carvedilol significantly increased beta-AR densities in lung membranes by 719% and 828%, respectively. (iii) Alprenolol, a beta-blocker with partial agonist properties at beta-ARs, behaved as a beta-AR agonist, and acutely reduced peak Raw value by 75.7% (P < 0.05); chronically, it did not alter Raw. (iv) Salbutamol, a beta-AR partial agonist, acutely decreased peak Raw by 41.1%; chronically, it did not alter Raw. (v) None of the beta-blockers produced significant changes in eosinophil number recovered in bronchoalveolar lavage. These results suggest that beta-AR agonists and beta-blockers with inverse agonist properties may exert reciprocating effects on cellular signaling dependent on duration of administration.

Effect of ??2-adrenergic receptor agonists on intercellular adhesion molecule (ICAM)-1, B7, and CD40 expression in mixed lymphocyte reaction

The plasma interleukin (IL)-18 level is elevated in acute rejection after organ transplantation. Although beta2-adrenergic receptor (AR) agonists suppress the rejection of organ and tissue transplants, little is known about their action mechanisms. We examined the effects of endogenous catecholamines and beta2-AR agonists on the expression of intercellular adhesion molecule (ICAM)-1, B7.1, B7.2, CD40, and CD40 ligand (CD40L) in human mixed lymphocyte reaction (MLR) and in an in vitro model of acute rejection in the presence or absence of IL-18. ICAM-1, B7.1 B7.2, CD40, and CD40L expression on monocytes was measured by flow cytometry, and the production of interferon (IFN)-gamma and IL-12 was determined by enzyme-linked immunosorbent assay. Lymphocytes proliferation in MLR was measured by [3H]-thymidine uptake. The relevant AR subtypes were characterized using subtype-selective agonists and antagonists. beta2-AR agonists inhibited the expression of ICAM-1 and CD40 during MLR in the absence of IL-18. Among IL-18-induced expression of ICAM-1, B7.1, B7.2, CD40, and CD40L, beta2-AR agonists inhibited ICAM-1 and CD40 expression. beta2-AR agonists prevented the production of IFN-gamma and IL-12 in the presence of IL-18 but had no effect in the absence of IL-18. beta2-AR agonists inhibited lymphocyte proliferation in IL-18-treated MLR. We found that beta2-AR agonists strongly inhibited the expression of ICAM-1 and CD40, irrespective of the presence or absence of IL-18, which is different from that of histamine and prostaglandin E2.

Quantitative estimation of myocardial fibrosis based on receptor occupancy for beta2-adrenergic receptor agonists in rats.

To develop beta2-adrenergic receptor (AR) agonists with higher selectivity, it is essential to evaluate the cardiac side effects which are the most serious side effects of this class of drugs. We studied receptor occupancy of beta1-ARs in rats as a possible cause for the side effect of beta2-AR agonists, namely myocardial fibrosis. Myocardial fibrosis in rats was observed on Day 7 after the administration of salbutamol and terbutaline, both of which are selective beta2-AR agonists, at higher dose levels. To evaluate receptor occupancy, plasma concentrations of (R)-salbutamol and (R)-terbutaline, plasma protein binding and the EC50 for chronotropic effects in rats were determined. Based on the plasma concentrations, the plasma protein binding and EC50, receptor occupancy-time profiles were constructed. The relationship between the receptor occupancy-time profile under the curve, the AUCphi, and the degree of myocardial fibrosis was evaluated with a multiple correlation analysis. Myocardial fibrosis was significantly correlated (r2 > 0.78) to the AUCphi with the threshold above approximately 50%, but not to plasma concentrations. These results indicate that the receptor occupancy theory is also useful for the evaluation of the chronotropic side effects of beta2-AR agonists.

Delayed but sustained induction of mitogen-activated protein kinase activity is associated with beta-adrenergic receptor-mediated morphological differentiation of astrocytes.

Astroglial beta-adrenergic receptors (beta-ARs) are functionally linked to regulate cellular morphology. In primary cultures, the beta-AR agonist isoproterenol (ISP) can transform flat polygonal astrocytes into process-bearing, mature stellate cells by 48 h, an effect that can be blocked by the beta-AR antagonist, propranolol. ISP induced immediate activation of protein kinase A (PKA) which persisted up to 2 h, with no visible change in cell morphology. However, activation of PKA was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. In addition to PKA inhibitors, the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 also blocked ISP-induced morphological transformation. ISP treatment resulted in a biphasic response of cellular phosphorylated MAPK (phosphorylated extracellular signal-regulated kinase; p-ERK) level: an initial decline in p-ERK level followed by a sustained induction at 12-24 h, both of which were blocked by PKA inhibitor. The induction in pERK level coincided with initiation of morphological differentiation of the astrocytes and nuclear translocation of p-ERK. A long-lasting activation of p-ERK activity by ISP, at a later stage, appears to be critical for the transformation of astrocytes.

Enhanced G(i) signaling selectively negates beta2-adrenergic receptor (AR)--but not beta1-AR-mediated positive inotropic effect in myocytes from failing rat hearts.

Myocardial contractile response to beta1- and beta2-adrenergic receptor (AR) stimulation is severely impaired in chronic heart failure, in which G(i) signaling and the ratio of beta2/beta1 are often increased. Because beta2-AR but not beta1-AR couples to G(s) and G(i) with the G(i) coupling negating the G(s)-mediated contractile response, we determined whether the heart failure-associated augmentation of G(i) signaling contributes differentially to the defects of these beta-AR subtypes and, if so, whether inhibition of G(i) or selective activation of beta2-AR/G(s) by ligands restores beta2-AR contractile response in the failing heart. Cardiomyocytes were isolated from 18- to 24-month-old failing spontaneously hypertensive (SHR) or age-matched Wistar-Kyoto (WKY) rat hearts. In SHR cardiomyocytes, either beta-AR subtype-mediated inotropic effect was markedly diminished, whereas G(i) proteins and the beta2/beta1 ratio were increased. Disruption of G(i) signaling by pertussis toxin (PTX) enabled beta2- but not beta1-AR to induce a full positive inotropic response in SHR myocytes. Furthermore, screening of a panel of beta2-AR ligands revealed that the contractile response mediated by most beta2-AR agonists, including zinterol, salbutamol, and procaterol, was potentiated by PTX, indicating concurrent G(s) and G(i) activation. In contrast, fenoterol, another beta2-AR agonist, induced a full positive inotropic effect in SHR myocytes even in the absence of PTX. We conclude that enhanced G(i) signaling is selectively involved in the dysfunction of beta2- but not beta1-AR in failing SHR hearts and that disruption of G(i) signaling by PTX or selective activation of beta2-AR/G(s) signaling by fenoterol restores the blunted beta2-AR contractile response in the failing heart.

Moderate weight-lowering effect of octopamine treatment in obese Zucker rats.

Octopamine is proposed as a substitution product of synephrine by diverse drug industries that advertise new weight-lowering products or medicinal plants enriched in this biogenic amine. We have already reported that octopamine is able to activate in vitro lipolysis in rat adipocytes via beta3-adrenergic receptor activation, while it activates glucose uptake in human fat cells via its oxidation by amine oxidases. In this work, we tested whether a chronic challenge with octopamine could exert anti-obesity effects. A treatment consisting in daily i.p. administration of octopamine (81 micromol/kg) was compared on a four-week period with calorie restriction in the genetically obese Zucker rat. Octopamine treatment resulted in a 19% decrease in body weight gain, when compared to the 177 g gained by controls during the same period. The decrease in body weight gain was detectable only after three weeks of treatment and was apparently not due to a pronounced and sustainable anorectic effect of octopamine since: 1) cumulated food consumption was only reduced by 10%; 2) the experimental 18% reduction of food intake provoked a rapid decrease in body weight gain, significant in less than two weeks. The lipolytic responses to isoprenaline or octopamine and the stimulation of glucose transport by insulin or by the amine oxidase substrate tyramine were unmodified by the treatments. Noteworthy, the elevated plasma insulin of obese rats was lowered by octopamine. This study shows that octopamine can reduce body weight gain in obese rats, without apparent adverse effects, but with less efficacy than beta3-AR agonists.

Gender- and site-related effects on lipolytic capacity of rat white adipose tissue.

Gender- and site-related differences in the lipolytic capacity, at the different steps of the adrenergic pathway, in gonadal and inguinal white adipose tissue (WAT), were assessed by studying alpha2A-adrenergic receptor (AR), beta3-AR and hormone-sensitive lipase (HSL) protein levels, and by determining the lipolytic response to different agents. Gonadal WAT showed a lower alpha2A/beta3-AR ratio, a greater lipolytic capacity in response to AR agonists, and higher HSL activity and protein levels than inguinal WAT. In female rats, we found greater alpha2A-AR protein levels and alpha2A/beta3-AR ratio compared to their male counterparts, but, on the other hand, a higher lipolytic response to beta-AR agonists and a greater lipolytic capacity at the postreceptor level, including a more activated HSL protein. Thus, the lipolytic capacity was clearly higher in gonadal than in inguinal WAT, at the different steps of the adrenergic pathway studied. Moreover, in both tissues, females showed a greater inhibition of lipolysis via alpha2-AR, which was counteracted by the higher lipolytic capacity at the postreceptor level.

Role of adrenergic receptor subtypes in the control of human placental blood vessels.

The use of beta2-Adrenergic Receptor (AR) agonists and the potential use of alpha1-AR blockers as tocolytics raise the question of how they influence placental circulation. The receptor profile was characterized via the amounts of the mRNA of alpha1-AR subtypes and beta2-ARs. The mRNAs were detected by reverse transcription-polymerase chain reaction. Electric field stimulation (EFS) was applied to test the pharmacological reactivity of the placental vessels. Expressions of beta2- and all subtypes of alpha1-AR mRNA were demonstrated in human placental vessels, and were significantly higher in the arteries. A significant difference was not found between the veins and the arteries as concerns the amount of alpha1D-AR mRNA. There was a preponderance of alpha1A- and alpha1B-AR mRNA as compared to alpha1D-AR mRNA both in the arteries and in the veins. beta2-AR agonists and alpha1-AR antagonists antagonized the EFS-induced contractions of the placental arteries in a dose-dependent manner. These effects were significantly less marked on veins at all applied doses. Urapidil antagonized the EFS-induced contractions of both the placental arterial and vein rings in a dose-dependent manner. The beta2-, alpha1A-, and alpha1B-AR are the important subtypes involved in the regulation of the contractility of the human term placental vessels. The possible increase in placental blood flow mediated by these ARs can even be beneficial during pregnancy. Accordingly, the use of beta2-AR agonists and the potential use of alpha1-blockers as tocolytics seem safe on the basis of in vitro examinations as concerns the placental circulation.

A Quantitative Analysis of mRNA Expression of α1 and β-adrenoceptor Subtypes and Their Functional Roles in Human Normal and Obstructed Bladders

We compared the expression level of alpha 1-adrenoceptor (AR) subtype mRNA to that of beta-AR subtype mRNA in control and obstructed human bladders, and examined whether alpha 1-AR mediated contraction and beta-AR mediated relaxation of human detrusor muscle are altered by bladder outlet obstruction. A real-time quantitative reverse transcriptase-polymerase chain reaction based method was used to quantify alpha1 and beta-AR subtype mRNA expression. The contractile response to alpha-AR agonists and the relaxant effect of beta-AR agonists were examined using an isometric contraction technique. The mRNA of alpha 1a, alpha 1b, alpha 1d, beta 1 and beta 2-AR mRNAs was expressed at low levels, while beta 3-AR was the most highly expressed subtype at the mRNA level in control and obstructed bladders. The expression level of alpha 1 and beta-AR subtype mRNA was not significantly different between the 2 groups. The mean contractile response to 10(-4) M phenylephrine +/- SEM was only 4.4% +/- 1.4% and 5.2% +/- 1.4% of the 10(-7) M carbachol induced contraction in the control and obstructed groups, respectively. Contractile responses to phenylephrine were not significantly increased in obstructed bladder. Isoproterenol and the beta 3-AR selective agonist L755,507 relaxed human detrusor muscle in a concentration dependent manner (10(-9) to 10(-4) M). However, the beta 1/beta 2-AR agonist dobutamine and the beta 2-AR selective agonist clenbuterol did not produce relaxation at a concentration of 10(-9) to 10(-5) M in the 2 groups. These findings indicate that neither up-regulation of alpha1-ARs nor down-regulation of beta-ARs occurs and relaxation mediating beta 3-ARs are by far predominant in the human obstructed bladder. Therefore, it is not likely that bladder alpha1-ARs are responsible for detrusor overactivity and storage symptoms in patients with benign prostatic obstruction.

Functional Characterization of β-adrenoceptor Subtypes in the Canine and Rat Lower Urinary Tract

We compared the effect of a beta 3-adrenoceptor (AR) agonist with that of beta 1 and beta 2-AR agonists on the urethra and bladder in the dog and rat. In an in vitro experiment we studied the relaxant effect of subtype selective beta-AR agonists in canine and rat urethral and bladder smooth muscle using an organ bath method. In addition, in urethane anesthetized rats we measured urethral pressure and bladder pressure simultaneously in the presence of the beta 3-agonist CL316243 and the beta 2-agonist procaterol in 4 or 5 animals. In the dog the relaxing effects of isoprenaline in the distal urethra were about half those seen in the detrusor and trigone. The rank order of relaxing potency was CL316243 > dobutamine (beta 1-agonist) = procaterol in detrusor and trigone but procaterol > dobutamine = CL316243 in the prostatic and distal urethra. In rat urethral smooth muscle in vitro the corresponding order was procaterol > CL316243 > dobutamine and the maximal relaxation to each agonist was about half that seen in the bladder. In the anesthetized rat procaterol clearly decreased urethral pressure but CL316243 produced only a slight decrease at its maximal dose, although each agonists clearly reduced bladder pressure. The beta 2-antagonist ICI-118551 counteracted the decrease in urethral and bladder pressure induced by procaterol. In rats and dogs a selective beta 3-AR agonist can decrease bladder pressure without affecting urethral pressure.

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.

Stimulation of औ2-Adrenergic Receptor Increases Cystic Fibrosis Transmembrane Conductance Regulator Expression in Human Airway Epithelial Cells through a cAMP/Protein Kinase A-independent Pathway

PSD-95/Dlg-A/ZO-1 (PDZ) domains play an essential role in determining cell polarity. The Na(+)/H(+) exchanger regulatory factor (NHERF), also known as EBP50, contains two PDZ domains that mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. Moreover, it has been shown that cystic fibrosis transmembrane conductance regulator (CFTR) and beta(2)-adrenergic receptor (beta(2)AR) bind equally well to the PDZ1 domain of EBP50. We hypothesized that beta(2)AR activation may regulate CFTR protein expression. To verify this, we evaluated the effects of a pharmacologically relevant concentration of salmeterol (2.10(-7) m), a long acting beta(2)AR agonist, on CFTR expression in primary human airway epithelial cells (HAEC). beta(2)AR stimulation induced a time-dependent increase in apical CFTR protein expression, with a maximal response reached after treatment for 24 h. This effect was post-transcriptional, dependent upon the beta(2)AR agonist binding to beta(2)AR and independent of the known beta(2)AR agonist-mediated cAMP/PKA pathway. We demonstrated by immunohistochemistry that CFTR, beta(2)AR, and EBP50 localize to the apical membrane of HAEC. Analyses of anti-EBP50 protein immunoprecipitate showed that salmeterol induced an increase in the amount of CFTR that binds to EBP50. These data suggest that beta(2)AR activation regulates the association of CFTR with EBP50 in polarized HAEC.

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.

Exercise restores beta-adrenergic vasorelaxation in aged rat carotid arteries.

Aging is associated with alterations in beta-adrenergic receptor (beta-AR) signaling and reduction in cardiovascular responses to beta-AR stimulation. Because exercise can attenuate age-related impairment in myocardial beta-AR signaling and function, we tested whether training could also exert favorable effects on vascular beta-AR responses. We evaluated common carotid artery responsiveness in isolated vessel ring preparations from 8 aged male Wistar-Kyoto (WKY) rats trained for 6 wk in a 5 days/wk swimming protocol, 10 untrained age-matched rats, and 10 young WKY rats. Vessels were preconstricted with phenylephrine (10-6 M), and vasodilation was assessed in response to the beta-AR agonist isoproterenol (10-10-3 x 10-8 M), the alpha2-AR agonist UK-14304 (10-9-10-6 M), the muscarinic receptor agonist ACh (10-9-10-6 M), and nitroprusside (10-8-10-5 M). beta-AR density and cytoplasmic beta-AR kinase (beta-ARK) activity were tested on pooled carotid arteries. beta-ARK expression was assessed in two endothelial cell lines from bovine aorta and aorta isolated from a 12-wk WKY rat. beta-AR, alpha2-AR, and muscarinic responses, but not that to nitroprusside, were depressed in untrained aged vs. young animals. Exercise training restored beta-AR and muscarinic responses but did not affect vasodilation induced by UK-14304 and nitroprusside. Aged carotid arteries showed reduced beta-AR number and increased beta-ARK activity. Training counterbalanced these phenomena and restored beta-AR density and beta-ARK activity to levels observed in young rat carotids. Our data indicate that age impairs beta-AR vasorelaxation in rat carotid arteries through beta-AR downregulation and desensitization. Exercise restores this response and reverts age-related modification in beta-ARs and beta-ARK. Our data support an important role for beta-ARK in vascular beta-AR vasorelaxation.

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.