Selective labelling of beta 1-adrenoceptors in rabbit lung membranes by (-)[3H]bisoprolol

The binding properties of a newly developed, highly selective beta 1-adrenoceptor antagonist radioligand, (-)[3H]bisoprolol (EMD 33512) were investigated in rabbit lung membranes containing a mixture of 80% beta 1-and 20% beta 2-adrenoceptors. The binding of (-)[3H]bisoprolol at 25 degrees C was saturable, of high affinity (KD = 4.7 +/- 0.6 nM, N = 4), rapid and readily reversible. The maximal number of (-)[3H]bisoprolol binding sites (244 +/- 31 fmol bound/mg protein, N = 4), however, was only 80% of the number of sites labelled by the non-selective beta-adrenoceptor radioligand (-)[125I]iodocyanopindolol (299 +/- 36 fmol bound/mg protein, N = 4). beta-Adrenoceptor antagonists (non-selective: propranolol, alprenolol; beta 1-selective: metoprolol, practolol, bisoprolol; beta 2-selective: ICI 118,551) inhibited (-)[3H]bisoprolol binding with monophasic displacement curves and pseudo-Hill coefficients of 1.0 indicating that in rabbit lung membranes (-)[3H]bisoprolol labels a homogeneous class of beta-adrenoceptors. Agonists inhibited binding with an order of potency: (-)-isoprenaline greater than (-)-noradrenaline = (-)-adrenaline, which is a typical one for beta 1-adrenoceptors. It is concluded that in rabbit lung membranes (-)[3H]bisoprolol selectively labels beta 1-adrenoceptors. (-)[3H]Bisoprolol therefore seems to be a suitable ligand for direct determination of the properties of beta 1-adrenoceptors in those tissues where both beta-adrenoceptor subtypes coexist.     

Selective regulation of beta 1- and beta 2-adrenoceptors in the human heart by chronic beta-adrenoceptor antagonist treatment.

1. In 44 patients undergoing coronary artery bypass grafting, the effect of chronic administration of the beta-adrenoceptor antagonists sotalol, propranolol, pindolol, metoprolol and atenolol on beta-adrenoceptor density in right atria (containing 70% beta 1- and 30% beta 2-adrenoceptors) and in lymphocytes (having only beta 2-adrenoceptors) was studied. 2. beta-Adrenoceptor density in right atrial membranes and in intact lymphocytes was assessed by (-)-[125I]-iodocyanopindolol (ICYP) binding; the relative amount of right atrial beta 1- and beta 2-adrenoceptors was determined by inhibition of ICYP binding by the selective beta 2-adrenoceptor antagonist ICI 118,551 and analysis of the resulting competition curves by the iterative curve fitting programme LIGAND. 3. With the exception of pindolol, all beta-adrenoceptor antagonists increased right atrial beta-adrenoceptor density compared to that observed in atria from patients not treated with beta-adrenoceptor antagonists. 4. All beta-adrenoceptor antagonists increased right atrial beta 1-adrenoceptor density; on the other hand, only sotalol and propranolol also increased right atrial beta 2-adrenoceptor density, whereas metoprolol and atenolol did not affect it and pindolol decreased it. 5. Similarly, in corresponding lymphocytes, only sotalol or propranolol increased beta 2-adrenoceptor density, while metoprolol and atenolol did not affect it and pindolol decreased it. 6. It is concluded that beta-adrenoceptor antagonists subtype-selectively regulate cardiac and lymphocyte beta-adrenoceptor subtypes. The selective increase in cardiac beta 1-adrenoceptor density evoked by metoprolol and atenolol may be one of the reasons for the beneficial effects observed in patients with end-stage congestive cardiomyopathy following intermittent treatment with low doses of selective beta 1-adrenoceptor antagonists.     

Lack of effect of chronic calcium antagonist treatment on beta 1- and beta 2-adrenoceptors in right atria from patients with or without heart failure.

1. We studied the effects of chronic calcium antagonist (calcium entry blocker, CEB; nifedipine, verapamil, diltiazem) treatment on beta-adrenoceptor density (assessed by (-)-[125I]-iodocyanopindolol [ICYP] binding) and subtype distribution in right atria from 65 patients without apparent heart failure undergoing elective coronary artery bypass grafting (CAD-patients) and from 13 patients with moderate heart failure (NYHA class III to class III-IV) undergoing mitral valve replacement (MVD-patients). 2. In CAD-patients atrial beta-adrenoceptor density was 79.3 +/- 7.9 fmol ICYP bound mg-1 protein (n = 18), the beta 1:beta 2-adrenoceptor ratio 69:31%. Chronic CEB-treatment did not affect either atrial beta-adrenoceptor density or beta 1:beta 2-adrenoceptor ratio. 3. In contrast, in CAD-patients chronically treated with beta 1-adrenoceptor antagonists (atenolol, bisoprolol, metoprolol) and CEB, atrial beta-adrenoceptor density was significantly increased (108.6 +/- 10.5 fmol ICYP bound mg-1 protein, n = 21); this increase was due to a selective increase in beta 1-adrenoceptors. 4. In MVD-patients atrial beta-adrenoceptor density (55.5 +/- 8.7 fmol ICYP bound mg-1 protein, n = 7) was significantly lower (P less than 0.05) than in CAD-patients; beta 1:beta 2-adrenoceptor ratio, however, was not changed (67:33%). Chronic CEB-treatment of MVD-patients did not prevent the decrease in atrial beta-adrenoceptors. 5. We conclude that chronic CEB-treatment does not affect human right atrial beta-adrenoceptor density, either in patients without apparent heart failure or in patients with moderate heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)     

FUNCTION, CHARACTERIZATION AND AUTORADIOGRAPHIC LOCALIZATION AND QUANTITATION OF β-ADRENOCEPTORS IN CARDIAC TISSUES

1. This paper demonstrates the use of organ bath, radioligand binding and autoradiography to detect beta 1- and beta 2-adrenoceptors in human and guinea-pig cardiac tissues. 2. In organ bath experiments, non-selective and beta 1- and beta 2-adrenoceptor selective agonists produced concentration-dependent inotropic responses in human right atrial appendage. Both subtypes mediate inotropic responses. In guinea-pig right atria chronotropic responses were mediated predominantly through beta 1-adrenoceptors. 3. Receptor binding studies using (-)[125I]-cyanopindolol (CYP) and beta 1- and beta 2-adrenoceptor selective antagonists showed that beta 2-adrenoceptors comprised 25% of the total population of beta-adrenoceptors in guinea-pig right atria. In human right atria the proportion is higher (40%). 4. Quantitative autoradiography was used to determine the location and densities of beta 1- and beta 2-adrenoceptors in guinea-pig heart. Both beta 1- and beta 2-adrenoceptors were distributed on myocardium. The atrioventricular conducting system had a higher density of beta 2-adrenoceptors compared with myocardium.     

Coexistence of beta 1- and beta 2-adrenoceptors in the rabbit heart: quantitative analysis of the regional distribution by (-)-3H-dihydroalprenolol binding

We determined the amount of beta 1- and beta 2-adrenoceptors in right and left atria and ventricles of rabbits. For this purpose inhibition of specific (-)-3H-dihydroalprenolol [(-)-3H-DHA] binding (5 nM) by beta 1-selective (practolol, metoprolol) and beta 2-selective (zinterol, IPS 339) adrenergic drugs was determined and analyzed by pseudo-Scatchard (Hofstee) plots. For both atria, inhibition of binding by the four selective beta-adrenergic drugs resulted in non-linear Hofstee plots, suggesting the coexistence of both beta-adrenoceptor subtypes. From these plots we calculated a beta 1:beta 2-adrenoceptor ratio of 72:28 for the right atrium and of 82:18 for the left. In contrast, only a very small amount of beta 2-adrenoceptors (approximately 5-7% of the total beta-adrenoceptor population) could be detected in the ventricles. For comparison we analyzed the inhibition of specific (-)-3H-DHA binding in tissues with homogeneous population of beta-adrenoceptors (beta 1:guinea pig left ventricle; beta 2: cerebellum of mature rats). For both tissues the four selective beta-adrenergic drugs showed linear Hofstee plots, demonstrating that in tissues with homogeneous beta-receptor population interaction of each drug with the receptor followed simple mass-action kinetics. We conclude that beta 1- and beta 2-adrenoceptors coexist in rabbit atria while the ventricles are predominantly endowed the beta 1-adrenoceptors.     

Characterization of beta 1- and beta 2-adrenoceptors in rat skeletal muscles.

Binding studies with (-)-[125I]cyanopindolol (ICYP) were conducted to characterize beta-adrenoceptors in plantaris and soleus muscles of rats (male, 250-300 g). The distribution of beta 1- and beta 2-adrenoceptors in different muscle fiber types, identified in serial sections by succinic dehydrogenase (SDH) staining, was studied by autoradiography. The densities of binding sites (Bmax, fmol/mg protein) were 5.4 +/- 0.9 (mean +/- SEM) in plantaris and 11.5 +/- 2.0 in soleus muscle. In plantaris muscle, monophasic competition curves were observed when binding experiments were performed using CGP20712A (50 pM to 0.5 mM), a beta 1-adrenoceptor selective antagonist, or ICI 118,551 (50 pM to 20 microM), a beta 2-adrenoceptor selective antagonist, to compete for ICYP binding. Analysis with LIGAND revealed a single binding site with a KD value of 2.41 +/- 0.56 nM (mean +/- SEM) for ICI 118,551 and 8.93 +/- 3.00 microM for CGP 20712A, indicating the presence of a homogeneous population of beta 2-adrenoceptors. In soleus muscle, competition curves were biphasic with 16-21% beta 1-adrenoceptors. Autoradiographic studies supported the findings from binding studies with membrane homogenates. The ICYP binding pattern was associated closely with the muscle fiber types identified by SDH staining. Propranolol-resistant binding sites were observed, and these sites were associated with muscle fibers positive to SDH staining.     

Beta-adrenoceptor antagonist activities and binding affinities of timolol enantiomers in rat atria

S-Timolol is an effective anti-glaucoma drug, but has potentially hazardous side effects. Recently, R-timolol, also, has been reported to be effective in lowering elevated intraocular pressure. In the present study, the beta-adrenoceptor antagonist activities and binding of R- and S-enantiomers of timolol have been examined on rat atrial preparations. The beta-antagonistic activities were investigated using spontaneously beating rat heart atria. Both timolol enantiomers inhibited (-)-isoprenaline-induced chronotropic action competitively. S-Timolol was about 54 times more potent than R-timolol. The apparent binding affinities of timolol enantiomers to beta 1- and beta 2-adrenoceptors were determined by a radioligand binding assay using (-)-[125I]iodocyanopindolol (ICYP) as a marker and CGP 20712 A as a beta 1- and ICI 118,551 as a beta 2-adrenoceptor antagonist. Both enantiomers of timolol inhibited ICYP binding in nanomolar concentrations with Hill coefficients near unity. Neither enantiomer showed selectivity between beta 1- and beta 2-adrenoceptors, but R-timolol was approximately 30 times less active than S-timolol. It is concluded that R-timolol is a relatively potent non-selective beta-adrenoceptor blocking agent, but may possibly exert a more localized beta-adrenoceptor action in the eye than S-timolol, thus improving the safety of ocular timolol therapy.     

Comparison of beta-adrenoceptor populations in cat and guinea-pig left atria

The subtypes of beta-adrenoceptor present in left atrial preparations from the guinea-pig and cat have been assessed using both responses obtained in organ bath experiments and radioligand binding studies. From the positive inotropic responses to procaterol, the pKB values for practolol using a variety of agonists, and from displacement of [125I]cyanopindolol from left atrial membrane homogenates by the selective beta 1- and beta 2-adrenoceptor antagonists L643,717-01J10 and ICI 118,551, it was concluded that guinea-pig left atria possess only beta 1-adrenoceptors, whilst cat left atria possess both beta 1- and beta 2-adrenoceptor subtypes.     

The affinity of betaxolol, a beta 1-adrenoceptor-selective blocking agent, for beta-adrenoceptors in the bovine trachea and heart.

1. The specificity of betaxolol, a beta-adrenoceptor antagonist, for beta 1- and beta 2-adrenoceptors was compared with that of other beta-antagonists, atenolol, ICI-118551, butoxamine and (+/-)-propranolol, in the bovine trachea and heart by competitive interaction with [3H]-CGP12177 as a radioligand. 2. The radioligand Kd values were 0.75 +/- 0.12 and 1.60 +/- 0.11 nM in the trachea and heart, respectively, and the Bmax values were 34.00 +/- 4.41 and 21.54 +/- 2.94 fmol mg-1 protein, respectively. 3. Using ICI-118551, we determined the ratio of beta 1:beta 2-adrenoceptors in the trachea and heart to be approximately 29:71 and 56:44, respectively. 4. In the trachea, a beta 2-predominant tissue, betaxolol and atenolol were more selective for beta 1-adrenoceptor binding sites than beta 2-adrenoceptor binding sites, whereas ICI-118551 and butoxamine were more selective for beta 2-adrenoceptor binding sites. 5. The beta 1-selectivity of betaxolol was 2.2 and 2.7 fold higher than that of atenolol in the bovine trachea and heart. These findings suggest that betaxolol may be useful in the treatment of hypertension, cardiac arrhythmia and angina pectoris.     

Pharmacological actions of the selective and non-selective beta-adrenoceptor antagonists celiprolol, bisoprolol and propranolol on human bronchi.

1. The pharmacological actions of the beta-adrenoceptor antagonists, celiprolol, bisoprolol and propranolol were investigated in human lung tissue by radioligand binding experiments as well as in human isolated bronchi by functional experiments in organ baths. 2. Data from lung tissue were compared to those obtained from myocardial membranes. 3. Lung tissue was obtained from 10 patients having undergone lung resection for bronchial carcinoma and myocardial tissue from a patient who had received a heart transplantation. 4. In radioligand binding experiments, celiprolol exhibited a high affinity binding to beta 1-adrenoceptors in heart and a low affinity binding to beta 2-adrenoceptors in lung tissue. The selectivity obtained for the beta 1-adrenoceptor was calculated to a factor of eleven. 5. Compared to bisoprolol and propranolol, celiprolol elicited the lowest affinity for the beta-adrenoceptor, as judged from the K1-values. 6. In the absence and presence of the guanine nucleotide Gpp(NH)p celiprolol did not affect receptor binding. 7. In functional experiments on intact bronchi, celiprolol, bisoprolol and propranolol failed to produce relaxation (+/- forskolin) or a significant difference in efficacy in antagonizing the relaxant effects of isoprenaline. However, a rank order of potencies was revealed (propranolol:bisoprolol:celiprolol = 46:12:1). 8. Plasma concentrations for celiprolol and bisoprolol usually achieved in vivo were below the IC50 value obtained in vitro. In contrast, for propranolol, plasma concentrations were nearly identical with the IC50 value. 9. It is concluded that celiprolol is a selective beta 1-adrenoceptor antagonist on human heart and has no agonistic properties on intact human bronchi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional beta1- and beta2-adrenoceptors in the left and right atrium of pre-hypertensive rats

There is a small increase in the functional beta2-adrenoceptor response on the spontaneously hypertensive rat (SHR) left atrium in the early stages of hypertension. In the present study, the functional beta1- and beta2-adrenoceptors of the left and right atrium in SHR pre-hypertension and age-matched (5-week-old) Wistar Kyoto (WKY) rats were characterized. Contractility methods with isoprenaline, T-0509 (a selective beta1-adrenoceptor agonist) and procaterol (a selective beta2-adrenoceptor agonist) were used. At 5 weeks, the SHRs were pre-hypertensive. Isoprenaline was more potent on the left atrium of 5-week-old SHRs than WKY rats. Bisoprolol, a selective beta1-adrenoceptor antagonist, was more potent against isoprenaline and T-0509 on the SHR than WKY rat left atrium. ICI 118,551, a selective beta(2)-adrenoceptor antagonist, was more potent against procaterol and T-0509 on the SHR than WKY rat left atrium. The results with bisoprolol and ICI 118,551 suggest that there are more functional beta(1)- and beta(2)-adrenoceptors on the left atrium of 5-week-old SHRs than WKY rats. Isoprenaline, T-0509 and procaterol were equipotent on the right atrium of 5-week-old WKY rats and SHRs. Bisoprolol was more potent against isoprenaline, T-0509 and procaterol on the SHR than WKY rat right atrium. ICI 118,551 was more potent against T-0509, but not isoprenaline and procaterol, on the SHR than WKY rat left atrium. This suggests there are more functional beta1-adrenoceptors, and probably more functional beta2-adrenoceptors, on the right atrium of 5-week-old SHRs than WKY rats. These functional differences in beta1- and beta2-adrenoceptor-mediated responses of the left and right atria of pre-hypertensive SHRs cannot be caused by hypertension, and may be associated with the onset of hypertension.     

Binding properties of beta-blockers at recombinant beta1-, beta2-, and beta3-adrenoceptors

The human heart contains at least four distinct beta-adrenoceptor subtypes, three of which have been cloned. However, the binding properties of beta-blockers to the different beta-adrenoceptor subpopulations are not yet thoroughly characterized. Human beta1-, beta2- and beta3-adrenoceptors were expressed in COS-7 cells and [125I]iodocyanopindolol saturation binding, and competition experiments with commonly used beta-blockers were performed in the respective membrane preparations. Atenolol and metoprolol were about fivefold selective for beta1- versus beta2- and beta3-adrenoceptors. Bisoprolol was approximately 15-fold selective for beta1- versus beta2- and approximately 31-fold selective for beta1- versus beta3-adrenoceptors. Carvedilol was nonselective for any beta-adrenoceptor subtype. We conclude that the beta1-selectivities of atenolol, metoprolol, and bisoprolol are lower in COS cell membranes compared with previous investigations performed in native membranes. All beta-blockers investigated bind to beta3-adrenoceptors. Differential binding properties to beta3-adrenoceptors might imply different responses as to body weight, cardiac contractility, heart rate, and growth regulation. This might imply differential indications for the drugs investigated.     

Blockade of beta 1- and desensitization of beta 2-adrenoceptors reduce isoprenaline-induced cardiac fibrosis

The aim of the present study was to analyse the role of beta(1)- and beta(2)-adrenoceptors in the catecholamine-induced myocardial remodeling, especially the interstitial fibrosis. Wistar rats were subjected to a 2-week chronic isoprenaline administration (30 microg/kg/h). Rats received a concomitant treatment with the selective beta(1)-adrenoceptor antagonist, bisoprolol (50 mg/kg/day p.o.) or were chronically pretreated with the selective beta(2)-adrenoceptor agonist salbutamol (40 microg/kg/h) for 1 week to induce beta(2)-adrenoceptor desensitization. The pretreatment with salbutamol induced a 59% down-regulation of left ventricular beta(2)-adrenoceptors compared to control. The extent of the isoprenaline-induced left ventricular fibrosis was significantly reduced in both the bisoprolol and salbutamol groups compared with the control isoprenaline-treated group especially in the apical region (1.7+/-0.6% and 1.4+/-0.3% versus 6.0+/-1.3%, respectively, P<0.005). beta(1)-adrenoceptor blockade and beta(2)-adrenoceptors down-regulation provided similar protection against isoprenaline-induced cardiac interstitial fibrosis suggesting that both beta-adrenoceptors are involved in such cardiac remodeling process.     

Factors controlling beta 1-adrenoceptor affinity and selectivity

A membrane preparation of the calf heart left ventricle has been used after identification and characterization, as a source of myocardial beta 1-adrenoceptor for radioligand binding studies. The displacement of specifically bound (-)-[3H]dihydroalprenolol by some beta-adrenoceptor ligands appeared to be pH-dependent, which could be related to the ionization characteristics of the compounds. Among the usually four ionic species of the ligand, present at physiological pH, the cation was shown to govern beta 1-adrenoceptor affinity. Furthermore, quantitative structure affinity relationships for the interaction with beta 1- and beta 2-adrenoceptors were established for the phenoxypropanolamines, a class of beta-adrenoceptor ligands. The N-isopropyl-oxypropanolamine side chain itself does not discriminate between beta 1- and beta 2-adrenoceptors, whereas aromatic substitution ortho to the side chain induces some beta 2-selectivity. Selectivity for myocardial beta 1-adrenoceptors is mainly obtained by aromatic substitution para to the side chain. This substitution pattern yields a decrease in beta 2-adrenoceptor affinity, far more pronounced than the decrease in beta 1-adrenoceptor affinity.     

Positive inotropic effects due to partial agonistic activity of the beta-adrenoceptor antagonist celiprolol following amplification of cAMP formation in failing human myocardium.

The present study investigated the effects of celiprolol a novel beta 1-antagonist with partial beta 2-agonist activity on the human failing heart. Experiments were performed on isolated electrically driven atrial and ventricular cardiac preparations and in membrane preparations from the left ventricles of nine patients (four with dilated cardiomyopathy; five with ischemic cardiomyopathy) undergoing cardiac transplantation for terminal heart failure. Celiprolol produced a negative inotropic effect in atrial and ventricular heart muscle. However, in the presence of forskolin--which activates the catalyst of the adenylate cyclase-or the cAMP phosphodiesterase inhibitor milrinone, celiprolol produced concentration-dependent positive inotropic effects and positive lusitropic effects. Experiments with the beta 1-and beta 2-selective antagonists CGP 207.12A and ICI 118.551, respectively, suggest that the positive inotropic response is mediated by beta 2-adrenoceptors. In radioligand binding experiments, a selectivity of 15.7 [-Gpp(NH)p] or 23.9 [+Gpp(NH)p] as judged from the Ki values--of binding to beta 2-adrenoceptors was measured in the failing human ventricular myocardium. Competition curves with celiprolol alone and in the presence of the guanine nucleotide Gpp(NH)p revealed no evidence for agonist activity at beta 1- or beta 2-adrenoceptors. It is concluded that amplification of the cAMP response is able to unmask partial agonist activity of celiprolol in the failing human heart at beta 2-adrenoceptors. The inotropic measurements are a more sensitive approach than radioligand binding studies. Whether the pharmacological profile of celiprolol will be useful in conditions like heart failure is questionable with respect to the potential downregulation of beta 2-adrenoceptors by its partial agonist activity.     

Tracheal relaxing effects and beta 2-selectivity of TA-2005, a newly developed bronchodilating agent, in isolated guinea pig tissues.

Tracheal relaxing effects and beta 2-selectivity of TA-2005 were investigated by functional experiments and radioligand binding assay in guinea pigs in comparison with those of other beta-agonists, isoproterenol, procaterol, formoterol and salbutamol. The relaxing activity of TA-2005 on histamine-induced contraction in the isolated trachea was most potent among the five agonists, and it was blocked by a beta 2-selective antagonist (ICI 118,551) but not by a beta 1-selective antagonist (bisoprolol). The potency of the relaxing effect was in the order of TA-2005 (pD2 = 9.79) greater than formoterol greater than procaterol greater than isoproterenol greater than or equal to salbutamol. The positive chronotropic effect of TA-2005 was similar to that of isoproterenol; and it was more potent than those of formoterol, procaterol and salbutamol in the isolated atria. The selectivity for tracheal muscle to atria of these agonists were in the order of procaterol greater than greater than or equal to formoterol greater than TA-2005 greater than salbutamol much greater than isoproterenol. A radioligand binding experiment using guinea pig lung and cardiac ventricle as beta 2- and beta 1-adrenoceptor sources, respectively, has also demonstrated that TA-2005 possesses extremely high affinity (IC50 = 1.04 nM) and selectivity (38-fold) to beta 2-adrenoceptors. By addition of GTP, the competition curve of [125I]iodocyanopindolol shifted rightward, indicating the agonist property. These results confirmed that TA-2005 is a highly beta 2-selective agonist that exerts a potent tracheal relaxing effect.     

Beta-adrenoceptor-binding studies of the cardioselective beta blockers bisoprolol, H-I 42 BS, and HX-CH 44 BS to heart membranes and intact ventricular myocytes of adult rats: two beta 1-binding sites for bisoprolol.

beta-Adrenoceptor binding of cardioselective drugs to intact ventricular myocytes was performed using [3H]CGP-12177, a hydrophilic non-beta 1/beta 2-selective antagonist radioligand. The beta-adrenoceptor density on the intact cardiomyocytes was about 2 x 10(5) molecules/cell. The beta 1-selective antagonists H-I 42 BS and HX-CH 44 BS competed for [3H]CGP-12177 binding sites on the ventricular myocytes in an essentially monophasic manner with Ki = 72.6 nM and Ki = 76.7 nM, respectively. This is in contrast to the results of binding data from heart membranes, where these beta 1-antagonists bind in a biphasic manner with about 30% of an additional low affinity site, presumably corresponding to the beta 2-adrenoceptor. The beta 1-selective antagonist bisoprolol revealed two binding sites at the heart membranes and at the myocytes as well with Ki(1) = 34.2 and 20.0 nM and Ki(2) = 3,014 and 918 nM, respectively. Our results suggest that viable adult rat ventricular myocytes may contain two beta 1-adrenoceptor binding sites exhibiting different affinities for bisoprolol, whereas beta 2-adrenergic receptors are completely absent.     

Respective degree of expression of beta 1-, beta 2- and beta 3-adrenoceptors in human brown and white adipose tissues.

1. The possible existence of a beta 3-adrenoceptor in human brown and white adipose tissues was investigated by mRNA expression and binding studies. 2. The relative amounts of beta 1-, beta 2- and beta 3-adrenoceptor mRNA, as determined by total RNA Northern blot analysis in newborn brown adipose tissue, were 28, 63 and 9% respectively of the total beta-adrenoceptor mRNA. 3. The beta 1/beta 2-adrenoceptors of human brown adipose tissue plasma membranes were characterized using [3H]-CGP 12177 as a ligand. Their Kd and Bmax values were 1.9 nM and 156 fmol mg-1 of membrane proteins, respectively. The beta 3-adrenoceptor was characterized by use of the new specific radioligand [3H]-SB 206606. The binding of this ligand was stereospecifically displaced by the active R,R- or the inactive S,S-enantiomer of BRL 37344 up to a concentration of about 10 microM. The Kd and Bmax values of the brown adipose tissue membrane beta 3-adrenoceptors were 87 nM and 167 fmol mg-1 of proteins, respectively. A low affinity [3H]-CGP 12177 binding site population was also detected in these membranes. 4. In human omental white adipose tissue, no beta 3-adrenoceptor mRNA could be detected in total RNA Northern blots and the beta 1-and beta 2-adrenoceptor mRNAs represented 9 and 91%, respectively of the total beta-adrenoceptor mRNA, and no specific binding of [3H]-SB 206606 could be measured.     

Regulation of beta-adrenoceptors by catecholamines in the rabbit skeletal muscle

(-)-[125I]Cyanopindolol (ICYP) was used to characterise beta-adrenoceptors in the gastrocnemius muscle of the rabbit. The binding of ICYP was saturable. The KI value for the selective beta 2-adrenoceptor antagonist, ICI 118551, was 0.16 +/- 0.01 nM (mean +/- S.E.M) and for the selective beta 1-adrenoceptor antagonist, metoprolol, was 300.1 +/- 33.3 nM. Hofstee plots for both antagonists were linear indicating the presence of homogeneous beta 2-adrenoceptors in the skeletal muscle of the rabbit. In gastrocnemius muscles obtained from rabbits chronically pretreated in vivo with vehicle (0.1% ascorbic acid), adrenaline (40-50 nmol.kg-1.h-1) or noradrenaline (80-100 nmol.kg-1.h-1) via osmotic mini-pumps implanted for 10 days, the Bmax values were 44 +/- 3, 25 +/- 4 (P less than 0.05) and 41 +/- 7 (fmol.mg-1 protein) and KD values were 7.3 +/- 0.9, 6.3 +/- 0.8 and 9.3 +/- 2.5 (pM), respectively. Thus pretreatment with the circulating hormone adrenaline but not noradrenaline down-regulated the number of skeletal muscle beta 2-adrenoceptors but did not influence the affinity.     

An analysis of the beta 2-adrenoceptor selectivity in three series of beta-adrenoceptor agonists

The aim of the study was to analyse the beta 2-adrenoceptor selectivity earlier found in two series of catecholamines and one series of resorcinolamines (Johansson et al. 1986). The affinity of the compounds was assessed in binding studies in preparations from the guinea-pig left heart ventricle (beta 1-adrenoceptors) and the soleus muscle (beta 2-adrenoceptors) using 3H-CGP-12177 as radioligand. Further, the activation of the adenylate cyclase by the compounds was studied in the same preparations. Selectivity quotients were obtained from both functional effects and from affinity and adenylate cyclase activating studies. There was a good correlation between the selectivity quotients obtained in these two ways. Tertiary butyl substitution on the amino nitrogen gave the highest beta 2-adrenoceptor selectivity in both the catechol and resorcinol series. In comparison with their isopropyl substituted analogues the beta 2-adrenoceptor selectivity of these compounds (KWD 2026 and terbutaline) was mainly due to a change in affinity for the beta 1- and beta 2-adrenoceptors and, to a lesser degree, a change in intrinsic efficacy.