No evidence for temperature-dependent changes in the pharmacological specificity ofβ 1- adrenoceptors

In rabbit lung membranes known to contain both beta 1- and beta 2-adrenoceptors it was studied whether changes in incubation temperature may affect binding characteristics and/or selectivity of beta 1- and beta 2-adrenoceptor drugs. For this purpose inhibition of binding of the highly specific beta-adrenoceptor radioligand (+/-)-125iodocyanopindolol (ICYP) by beta 1-and beta 2-selective as well as non-selective drugs was determined at incubation temperatures of 37 degrees C and 18 degrees C and analyzed by Hofstee-plots. 1. The density of beta-adrenoceptors in rabbit lung membranes was identical independently of the incubation temperature. 2. At both incubation temperatures beta 1- and beta 2-selective drugs showed biphasic displacement curves and non-linear Hofstee-plots, while inhibition of binding by the non-selective drugs resulted in monophasic displacement curves with linear Hofstee-plots. With decreasing temperature affinity of antagonists to beta-adrenoceptors increased only slightly, while affinity of agonists increased markedly. 3. For all beta 1- and beta 2-selective drugs the same ratio beta 1/beta 2-adrenoceptors was calculated from the Hofstee-plots independently of the incubation temperature: It amounted to about 80% beta 1- and 20% beta 2-adrenoceptors in rabbit lung. 4. At an incubation temperature of 37 degrees C the displacement curve of the agonist isoprenaline was biphasic in the absence of GTP with a non-linear Hofstee-plot indicating that at 37 degrees C isoprenaline binds to high and low affinity states of the beta-adrenoceptors in rabbit lung. At 18 degrees C, however, beta-adrenoceptors do not form the high affinity GTP-sensitive complex with agonists, since GTP had no influence on isoprenaline displacement curves. 5. It is concluded that a decrease in the incubation temperature of ICYP binding assay from 37 degrees C to 18 degrees C does neither alter the relative amount of beta 1- and beta 2-adrenoceptors in rabbit lung membranes nor the selectivity of beta 1- and beta 2-selective adrenoceptor drugs.     

Regional distribution of beta-adrenoceptors in the human heart: coexistence of functional beta 1- and beta 2-adrenoceptors in both atria and ventricles in severe congestive cardiomyopathy

We evaluated the amount of beta 1- and beta 2-adrenoceptors in human right and left atrium as well as in right and left ventricular wall obtained from heart transplant recipients who suffered from end-stage congestive cardiomyopathy. The total number of myocardial beta-adrenoceptors was assessed with the nonsubtype selective beta-adrenoceptor radioligand (-)[125I]iodocyanopindolol (ICYP); concomitantly, the number of beta 1-adrenoceptors was determined with the selective beta 1-adrenoceptor radioligand (-)[3H]bisoprolol. The number of beta 2-adrenoceptors was calculated by subtracting (-)[3H]bisoprolol binding sites from ICYP binding sites. With this technique, a beta 1/beta 2-ratio of approximately 65/35% for both atria and of approximately 75/25% for both ventricles was found. Identical results were obtained when the beta 1/beta 2-ratio was calculated indirectly by nonlinear regression analysis of competition curves of the selective beta 1-adrenoceptor antagonist bisoprolol and the selective beta 2-adrenoceptor antagonist ICI 118,551 with ICYP binding. In addition, on atria and on ventricles, adenylate cyclase was activated by norepinephrine (presumably by beta 1- and beta 2-adrenoceptor stimulation) and by procaterol (by beta 2-adrenoceptor stimulation). It is concluded that in the human heart functional beta 1- and beta 2-adrenoceptors coexist on both atria and both ventricles. In end-stage congestive cardiomyopathy, there appears to be a selective down-regulation of cardiac beta 1-adrenoceptors, whereas beta 2-adrenoceptors are obviously not affected. This may explain the beneficial effects of beta 2-adrenoceptor agonists in severe heart failure.     

Characterization of beta-adrenoceptors on rat skeletal muscle cells grown in vitro

The binding properties of an hydrophilic beta-adrenergic receptor radioligand, (-)[3H](4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazolo-2-one ); ([3H]CGP-12177), were investigated in rat skeletal muscle cells in culture. The binding of [3H]CGP-12177 at 25 degrees was saturable, reversible and of high affinity (Kd = 1.3 +/- 0.3 nM). The maximal number of [3H]CGP-12177 binding sites was 30.6 +/- 3.2 fmol/dish (34 +/- 3.5 fmol/mg protein). beta-Adrenergic agonists and antagonists inhibited [3H]CGP-12177 binding. The competing ligand inhibition binding is a typical one for beta 2-adrenoceptors. The increase in beta-adrenoceptors was independent of cell fusion. Amiodarone (10(-5) M) decreased the beta-adrenoceptor number in skeletal muscle cells differentiated in vitro by 48%, while the affinity for [3H]CGP-12177 was not affected.     

Autoradiographic localization of beta-adrenoceptors in pig lung using [125I]-iodocyanopindolol.

The binding of the beta-adrenoceptor radioligand [125I]-iodocyanopindolol (I-CYP) has been studied in pig lung parenchyma and the distribution of binding sites visualised by light microscopic autoradiography. I-CYP binding was saturable (maximum binding capacity Bmax = 51 +/- 3 fmol mg-1 protein), involving sites with high affinity (dissociation constant KD = 73 +/- 10 pM). Specific I-CYP binding was displaceable both by beta-adrenoceptor agonists ((-)-isoprenaline greater than (-)-adrenaline greater than (+/-)-fenoterol greater than (-)-noradrenaline greater than (+)-isoprenaline greater than (+/-)-RO363) and antagonists ((+/-)-propranolol greater than ICI-118551 greater than atenolol), indicating a predominance of beta 2-adrenoceptors. Further analysis showed that displacement data for the beta 1-selective antagonist atenolol and the beta 2-selective antagonist ICI-118551 were fitted best to a 2 binding site model and that both beta 1- and beta 2-adrenoceptors were present in pig lung in the ratio 28:72 respectively. Autoradiographic grains were localized over tissue and were most dense over alveolar walls greater than vascular endothelium greater than vascular smooth muscle greater than bronchial smooth muscle = bronchial epithelium. Atenolol (10(-5) M) caused a 31% reduction in specific grain density over alveolar wall tissue, while a 10 fold lower concentration of ICI-118551 (10(-6) M) caused a 50% decrease. These results are consistent with binding data in pig lung parenchyma demonstrating a mixed population of beta-adrenoceptors with a predominance of the beta 2 subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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)     

Distribution of beta 1- and beta 2-adrenoceptors in mouse trachea and lung: a quantitative autoradiographic study.

1. Binding and quantitative autoradiography were used to detect [125I]-iodocyanopindolol (I-CYP) associated with beta 1- and beta 2-adrenoceptors in mouse tracheal epithelium and airway smooth muscle as well as in lung parenchymal tissue. 2. Specific I-CYP binding to slide-mounted tissue sections of both trachea and parenchyma was of high affinity (KD = 49.0 pM, n = 3, trachea; KD = 118.9 pM, n = 3, parenchyma) and saturable, involving single populations of non-interacting binding sites (Hill coefficient nH = 1.00 +/- 0.02, trachea; nH = 0.99 +/- 0.03, parenchyma). 3. Direct measurement of tissue radioactivity also showed that specific I-CYP binding was competitively inhibited in the presence of the beta-adrenoceptor antagonists (-)-propranolol (non-selective), CGP 20712A (beta 1-selective) and ICI 118,551 (beta 2-selective). Analysis of the competition binding curves for the two selective antagonists revealed mixed populations of beta 1- and beta 2-adrenoceptors in the approximate proportions 33% and 67% respectively in mouse trachea and 28% and 72% respectively in mouse lung parenchyma. 4. Densities of autoradiographic grains derived from specific I-CYP binding to alveolar wall tissue and to tracheal epithelium and airway smooth muscle were quantified by a computer-assisted image analysis system, which allowed the construction of competition binding curves in the presence of the selective beta-adrenoceptor antagonists CGP 20712A and ICI 118,551. Analysis of these data demonstrated that in alveolar wall, beta 1- and beta 2-adrenoceptors co-existed in the proportions 18% and 82%, respectively. 5. Quantitative autoradiographic analyses also showed that beta 1- and beta 2-adrenoceptors were differentially distributed in tracheal epithelium and airway smooth muscle. The beta 2-adrenoceptor subtype accounted for 71% of all beta-adrenoceptors in epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of beta-adrenoceptors in rat lung parenchymal strip

The aim of the present study was to characterize the beta-adrenoceptor population in rat lung strip. For this purpose, Schild plots were obtained for the beta-adrenoceptor antagonists atenolol (beta 1-selective), butoxamine (beta 2-selective) and propranolol (non-selective), using three different agonists:isoprenaline (non-selective), salbutamol (beta 2-selective) and noradrenaline (beta 1-selective). The slopes of these Schild plots were close to the theoretical value of unity, and pA2 values determined with isoprenaline, salbutamol and noradrenaline as agonists were: for propranolol, 7.86 +/- 0.22, 7.72 +/- 0.15 and 7.89 +/- 0.23; for atenolol, 5.19 +/- 0.05, 5.33 +/- 0.07 and 5.47 +/- 0.22 and for butoxamine, 6.31 +/- 0.11, 6.34 +/- 0.03 and 5.99 +/- 0.23, respectively. These data suggest that pharmacological responses of rat isolated lung strip to beta-adrenoceptor agents are mediated by a homogeneous population of beta 2-adrenoceptors, although the presence of a minor population of beta 1-adrenoceptors undetected by the agonists used cannot be excluded.     

[3H]-idazoxan binds with high affinity to two sites on hamster adipocytes: an alpha 2-adrenoceptor and a non-adrenoceptor site.

1. [3H]-idazoxan labels a single population of high affinity sites (Kd 2.26 +/- 0.02 nM; Bmax 372 +/- 25 fmol mg-1 protein) in hamster adipocyte membranes. In the presence of 1 microM yohimbine to preclude binding to alpha 2-adrenoceptors, the density of [3H]-idazoxan binding sites was reduced (287 +/- 18 fmol mg-1 protein) without an apparent decrease in the affinity (Kd 2.19 +/- 0.24 nM) of the radioligand. 2. Displacement studies indicate that alpha-adrenoceptor ligands with an imidazoline side chain completely inhibit [3H]-idazoxan binding to hamster adipocyte membranes; in contrast, the alpha 2-adrenoceptor antagonists yohimbine, rauwolscine, BDF 6143 and phentolamine inhibited only 20-30% of the specific binding with affinity values consistent with an interaction at alpha 2-adrenoceptors. 3. The low potency of noradrenaline and adrenaline in displacing [3H]-idazoxan binding to the second site on hamster adipocyte membranes indicates that it is unlikely that this site is a type of adrenoceptor. 4. These results suggest that [3H]-idazoxan binds with high affinity to two sites in hamster adipocytes: an alpha 2-adrenoceptor and a non-adrenoceptor imidazoline site.     

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.     

Beta 1-adrenoceptor selectivity of nebivolol and bisoprolol. A comparison of [3H]CGP 12.177 and [125I]iodocyanopindolol binding studies

There is an ongoing discussion on whether or not high beta(1)-adrenoceptor selectivity of beta-adrenoceptor antagonists may be favorable in the treatment of patients with heart failure. The present study compared the beta(1)-adrenoceptor selectivity of nebivolol and bisoprolol with that of carvedilol in the human myocardium, using a binding assay in conjunction with either the hydrophilic ligand (+/-)-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on HCl ([3H]CGP 12.177) or the lipophilic ligand [125I]iodocyanopindolol as radiolabeled compound. Measurements were made using membrane preparations obtained from identical nonfailing donor hearts. beta-adrenoceptor density was found to be slightly higher when [125I]iodocyanopindolol was used compared to [3H]CGP 12.177 (256+/-15 and 213+/-18 fmol/mg protein, respectively). When the highly beta(1)-adrenoceptor-selective compound 2-hydroxy-5-(2-(hydroxy-3-(4((1-methyl-4-trifluoromethyl)-1-H-imidazol-2-yl)-phenoxy)-propyl)-aminoethoxyl)-benzamide (CGP 20.712A) and the highly beta(2)-adrenoceptor-selective compound erythro-(+/-)-1-(7-methylindan-4-yloyl)-3-isopropylaminobutan-2-ol HCl (ICI 118.551) were used in competition experiments, a similar proportion of beta(1)-adrenoceptors was seen for [3H]CGP 12.177 (69.3+/-1.6%) and for [125I]iodocyanopindolol (67.0+/-2.1%). K(i)(beta(1)) and K(i)(beta(2)) were obtained in the presence of 50 nM ICI 118.551 and 300 nM CGP 20.712A. The rank order of beta(1)-adrenoceptor selectivity (K(i)(beta(2))/K(i)(beta(1)) ratio) was nebivolol (for [3H]CGP 12.177 46.1 and for [125I]iodocyanopindolol 22.5)>bisoprolol (13.1 and 6.4)>carvedilol (0.65 and 0.41). To investigate whether in vivo metabolized nebivolol retains high beta(1)-adrenoceptor selectivity, serum specimens were collected before and 2 h after oral administration of 5 mg nebivolol. The samples were used for [125I]iodocyanopindolol binding studies with the myocardial membrane preparations. In these samples, the binding of [125I]iodocyanopindolol to beta(1)-adrenoceptors was inhibited by 46.4+/-5.3%, whereas the binding to beta(2)-adrenoceptors was inhibited by 20.5+/-1.1% compared to that of control samples. It is concluded that nebivolol is approximately 3.5 times more beta(1)-adrenoceptor-selective than bisoprolol in the human myocardium. Furthermore, in vivo metabolized nebivolol retains beta(1)-adrenoceptor selectivity.     

3H]idazoxan binding at non-alpha 2-adrenoceptors in rabbit adipocyte membranes

The imidazoline ligand, [3H]idazoxan, labels a large population of high-affinity binding sites in rabbit fat cell membranes (Bmax = 1370 +/- 91 fmol/mg protein; KD = 1.6 +/- 0.6 nM) when imidazoline derivatives are used for definition of non-specific binding. [3H]Idazoxan sites are not alpha 2-adrenoceptors as assessed by competition studies which showed that epinephrine, norepinephrine and yohimbine do not inhibit [3H]idazoxan binding. Naphazoline, tramazoline and the Na+/H+ exchange inhibitor, amiloride, completely inhibited [3H]idazoxan binding. The Ki values were 9, 27 and 48 nM, respectively.     

Beta-adrenoceptors and human skeletal muscle characterisation of receptor subtype and effect of age.

1. Rectus abdominis muscle biopsies were obtained from 28 patients undergoing abdominal surgery. In membranes prepared from these biopsies beta-adrenoceptor binding was examined. The apparent affinity (KD) and the density (Bmax) of the receptors for the radioligand (-)-[125I]cyanopindolol were 28.5 +/- 2.7 (pM) and 25.9 +/- 2.1 (fmol mg-1 protein) (mean +/- s.e. mean) respectively. In forceps biopsies from vastus lateralis muscle from four healthy volunteers the values for KD and Bmax were 22.5 +/- 4.4 (pM) and 16.4 +/- 2.2 (fmol mg-1 protein). The binding characteristics for the radioligand were similar in the biopsies from the two muscle sites. 2. Inhibition of the radioligand binding by the selective beta 2-adrenoceptor antagonist ICI 118551 (KI = 117 +/- 45 nM) and selective beta 1-adrenoceptor antagonist metoprolol (KI = 15229 +/- 5046 nM) suggests the dominance of beta 2-adrenoceptor subtype in human skeletal muscle. 3. There were no significant differences in the skeletal muscle beta-adrenoceptor densities or affinities between the young and older patients.     

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)     

LK 204-545, a highly selective beta1-adrenoceptor antagonist at human beta-adrenoceptors

LK 204-545 ((+/-)-1-(2-(3-(2-cyano-4-(2-cyclopropyl-methoxy-ethoxy)phenoxy)-2-hydro xy-propyl-amino)-ethyl)-3-(4-hydrxy-phenyl) urea), an antagonist that possesses high beta1-/beta2-selectivity in the rat, and a range of cardio-selective and non-selective beta-adrenoceptor antagonists were examined to compare their radioligand binding affinities for human beta1-, beta2- and beta3-adrenoceptors transfected into CHO cells. LK 204-545 and CGP 20712A displayed the highest beta1-/beta2- (approximately 1800 and approximately 650, respectively) and beta1-/beta3-selectivity (approximately 17000 and approximately 2200, respectively) at human beta-adrenoceptors with LK 204-545 being approximately 2.75-fold more beta1-/beta2-selective and approximately 8-fold beta1-/beta3-selective than CGP 20712A. The high potency of LK 204-545 at transfected human beta1-adrenoceptors and in functional models of rat beta1-adrenoceptors together with its high selectivity, identify it as a useful ligand for studying beta1-adrenoceptors and suggest that it may be the preferred ligand for human beta-adrenoceptor studies.     

Pharmacological and biochemical characterization of the beta-adrenergic signal transduction pathway in different segments of the respiratory tract

Although in the respiratory system there is great therapeutic interest in manipulating and understanding the beta-adrenoceptor-G-protein-adenylate cyclase (AC) signal transduction pathway, little is known on segmental differences among lung, bronchus, and trachea with regard to the receptor concentration and interaction to G-proteins and coupling to AC. In this study, patterns of distribution and absolute quantities of beta-adrenoceptor subtypes beta(1) and beta(2) were determined in membranes of equine lung parenchyma, bronchial and tracheal epithelium with the underlying smooth muscle by saturation and competition binding assays using the radioligand (-)-[125I]-iodocyanopindolol (ICYP). Additionally, the functional coupling of beta-adrenoceptors to G-proteins (assessed by beta-agonist competition binding in the presence and absence of GTP) as well as the coupling efficiency and biochemical activities of AC was investigated in each region. The specific ICYP binding was rapid, reversible, saturable with time and of high affinity. The radioligand binding identified more total beta-adrenoceptors in the lung than in bronchus or trachea (428+/-19, 162.4+/-4.8, 75.6+/-1.2 fmol/mg protein, respectively) with about 40% of receptors in the high affinity state. The beta(2)-adrenoceptor subtype predominated in all segments (approximately 74-80%), as the highly selective beta(2)-adrenoceptor antagonist ICI 118,551 was about 10,000 times more potent in inhibiting ICYP binding than was the beta(1)-selective adrenoceptor antagonist CGP 20712A, and beta-adrenoceptor agonists inhibited ICYP binding with an order of potency: (-)-isoprenaline>(-)-adrenaline>(-)-noradrenaline. The dissociation constant (K(d)) was higher in the trachea than in bronchus or lung (13.0+/-0.9 pM vs. 20.0+/-2.3 pM vs. 30.8+/-4.4 pM, P<0.05, respectively). The beta(2)-adrenoceptor-mediated AC response was tissue-dependent; stimulants acting on beta-adrenoceptor (isoproterenol), G-protein (GTP, NaF) and AC (forskolin, Mn(2+)) enhanced AC responses in all three regions, but the AC activity was higher in tracheal crude membranes than in bronchus or lung (trachea>bronchus>lung), hence, the number of beta(2)-adrenoceptors correlated inversely with the amount of AC. We conclude that (1) the stoichiometry of components within the pulmonary beta-adrenoceptor-G-protein complex is segment-dependent, and (2) the receptor number or AC activity is possibly the rate-limiting factor in the beta-adrenoceptor-G-protein-AC-mediated physiological responses. Thus, it is speculated that this could have important therapeutic consequences in beta-adrenoceptor agonist-induced receptor regulation in bronchial asthma.     

Classical and atypical binding sites for beta-adrenoceptor ligands and activation of adenylyl cyclase in bovine skeletal muscle and adipose tissue membranes.

1. The radioligand [125I]-iodocyanopindolol ([125I]-ICYP) was used under standard ligand binding conditions, to detect beta 1- and beta 2-adrenoceptors in membrane preparations from bovine skeletal muscle and adipose tissue. High concentrations of [125I]-ICYP were also used, to identify an 'atypical' binding site in skeletal muscle. Finally, adenosine 3':5'-cyclic monophosphate (cyclic AMP) production was measured in the same membrane preparations, to determine the relationship between the beta-adrenoceptor sub-types present and the production of this second-messenger. 2. According to the results of radioligand binding studies, both skeletal muscle and adipose tissue membranes have beta 2-adrenoceptors, characterized by a high affinity for the beta 2-selective antagonist, ICI 118551 (pK 8.3 and 8.6 respectively); and a low affinity for the beta 1-selective antagonist CGP 20712A (pK 5.2 in both tissues). Antagonism of (-)-isoprenaline-stimulated cyclic AMP production by low concentrations of ICI 118551, yielded pseudo pA2 values in muscle and adipose tissue of 7.6 and 8.7 respectively, confirming that beta 2-adrenoceptors in these tissues are linked to the production of the second-messenger. 3. Although beta 1-adrenoceptors could not be detected in either skeletal muscle or adipose tissue membranes by use of ligand binding techniques, high pseudo pA2 values were obtained (8.0 and 8.2 respectively), when CGP 20712A was used to block the stimulation of cyclic AMP production by (-)-isoprenaline. This finding is consistent with the presence in both tissues of a population of beta 1-adrenoceptors which is small, but efficiently coupled to the second-messenger.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific and non-specific effects of beta-adrenoceptor agonists on guinea pig alveolar macrophage function

The existence of beta-adrenoceptors on guinea pig alveolar macrophage membranes was determined by means of radioligand binding studies. Saturable binding with [125I]cyanopindolol demonstrated 38 +/- 6 fmol binding sites per 10(6) alveolar macrophages with a Kd of 0.85 +/- 0.15 nM. With timolol, atenolol and ICI 118.551 for competition of [125I]cyanopindolol binding it became clear that guinea pig alveolar macrophages possessed adrenergic binding sites of the beta 2-subtype. The cyclic AMP levels of alveolar macrophages could be increased by selective beta 2-adrenoceptor agonists but not by selective beta 1-adrenoceptor agonists. The influence of non-selective beta- and selective beta 1- and beta 2-adrenoceptor agonists on the phagocytic and metabolic responsiveness of alveolar macrophages was also studied. Addition of beta-adrenoceptor agonists had no effect on the uptake of bacteria by alveolar macrophages. Incubation of alveolar macrophages with increasing amounts of non-selective and selective beta 1-agonists resulted in a dose-dependent decrease in the detection of hydrogen peroxide released by alveolar macrophages. This effect was due to the scavenging properties of these drugs. The selective beta 2-receptor agonists, salbutamol and terbutaline, had no effect on the oxidative metabolism of alveolar macrophages. We conclude that guinea pig alveolar macrophages possess beta 2-adrenoceptors on their cell surface and that these receptors are not involved in the phagocytic activity of alveolar macrophages.     

Ligand binding properties of putative beta 3-adrenoceptors compared in brown adipose tissue and in skeletal muscle membranes.

1. The beta-adrenoceptor population was characterized in membrane preparations from rat brown adipose tissue (BAT) and from soleus muscle by use of the radioligand [125I]-iodocyanopindolol ([125I]-ICYP). In addition, atypical binding sites for [125I]-ICYP found in both tissues were examined, and the relationship between these sites and the putative rat beta 3-adrenoceptor is discussed. 2. It was established that BAT membranes host a mixed population of beta 1- and beta 2-adrenoceptors. Of these two sites, 55% showed a high affinity for the beta 1-selective ligand CGP 20712A (pK 8.5), and 45% showed a high affinity for the beta 2-selective antagonist ICI 118551 (pK 8.6). Soleus muscle membranes were found to host a population of beta 2-adrenoceptors, characterized by a high affinity for ICI 118551 (pK 9.1), but beta 1-adrenoceptors could not be detected in this preparation. 5-Hydroxytryptamine receptors were not detected in either preparation. 3. In addition to beta 1- and beta 2-adrenoceptors, atypical binding sites were identified in both tissues using high concentrations of radioligand (0.5-0.6 nM) and in the presence of 1 microM (-)-propranolol. The atypical sites were abundant, representing 80 and 81% of the total [125I]-ICYP binding sites in BAT and soleus muscle respectively. When the pK values for 11 ligands were compared, the correlation coefficient for atypical sites in BAT and soleus muscle was 0.94.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of cerebral beta-adrenoceptor binding sites in various vertebrate species.

[3H]-Dihydroalprenolol ([3H]-DHA) binds to cerebral membranes of the frog, chick, rat, mouse, rabbit and human with a dissociation equilibrium constant (KD) of about 1 nM and displays binding characteristics indicative of an interaction with beta-adrenoceptors. However, the maximum number of specific binding sites labelled by this beta-adrenoceptor ligand varies substantially between the species with the chick and mouse having the highest, and the frog the lowest density. The structure--activity relationships of adrenergic agents to inhibit specific [3H]-DHA binding suggests that whereas the membrane sites from all the species had similar affinities for non-selective beta-adrenergic agents, several drugs that have been reported to show selectivity for beta1-adrenoceptors demonstrated considerably higher affinities for mammalian rather than avian or amphibian membrane sites. By this pharmacological criteria it is likely that all the beta-adrenoceptor binding sites in frog and chick cerebral tissue have properties resembling beta2-receptors. However, in mammalian cerebral cortex, evidence is presented that beta1- and beta2-adrenoceptors coexist in a ratio of 70%/30% respectively.