Binding of beta-adrenoceptor antagonists to rat and rabbit lung: special reference to levobunolol

Binding of 3H-dihydroalprenolol (3H-DHA) to beta-adrenoceptors in homogenates from rat and rabbit lung was homogeneous and of high affinity (KD = 0.6 and 1.1 nmol/l at 20 degrees C; 1.1 and 2.2 nmol/l at 37 degrees C). The beta 1-selective antagonists betaxolol, metoprolol, bevantolol and acebutolol displaced 3H-DHA in a biphasic manner. From these data, the beta-adrenoceptor subtype distribution in rat lung homogenates was estimated to be 80% beta 2 (at 20 and 37 degrees C) as compared to 25% beta 2 in rabbit lung homogenates. In general, binding of beta-adrenoceptor antagonists (selective and nonselective) was slightly (less than 2 X) weaker in rabbit than in rat lung homogenates. In rat lung, binding of cardioselective beta-blockers to beta 1-receptors seemed to be more temperature-sensitive than binding to beta 2-receptors or binding of nonselective beta-blockers. Levobunolol, a potent non-cardioselective beta-blocker in pharmacological experiments, displaced 3H-DHA in a homogeneous manner (indicative of non-selectivity). In rat lung homogenates KD values were 0.8 nmol/l at 20 degrees C and 2.1 nmol/l at 37 degrees C. Similar values were found for the metabolites dihydrolevobunolol and hydroxylevobunolol. Surprisingly, d-bunolol, the dextrarotatory enantiomer of bunolol, showed a biphasic displacement curve, the fraction of high affinity sites being 83% in rat lung homogenates and 23% in rabbit lung. This ratio of sites is expected for a beta 2-adrenoceptor preferring ligand. High affinity binding (i.e. supposedly binding to beta 2-receptors) was about 50 times weaker than binding of levobunolol, in agreement with known stereospecificity of beta-adrenoceptor binding.     

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.     

Effects of bunitrolol on adrenergic and serotonergic receptors

To assess the importance of anti-adrenergic and anti-serotonergic activities of bunitrolol for its efficacy as an antihypertensive and antianginal agent, effects of this substance on the binding of adrenergic and serotonergic agents to the respective receptors of the rat brain, rat heart, dog brain, and/or dog aorta were examined using the radioligand binding assay methods. In addition, the pA2 values of bunitrolol as an antagonist against the positive chronotropic and inotropic actions (beta 1-adrenoceptor) of isoproterenol were also determined by pharmacological methods using the isolated guinea pig atria. To assess the specificity, pA2 values were also obtained in the isolated trachea (beta 2-adrenoceptor) using isoproterenol as an agonist and in the isolated aorta from the guinea pig and the rat using phenylephrine as an agonist (alpha 1-adrenoceptor). A strong inhibition by bunitrolol of 3H-dihydroalprenolol (3H-DHA) binding to beta-adrenoceptors was observed, while the inhibition of 3H-prazosin binding to alpha 1-adrenoceptors, 3H-serotonin binding to 5HT1-receptors. 3H-p-aminoclonidine binding to alpha 2-adrenoceptors, and 3H-ketanserin binding to 5HT2-receptors were found to be very weak. The rank order of antagonistic potencies of bunitrolol against the adrenergic receptors as assessed with pA2 values were beta 1 greater than beta 2 much greater than alpha 1. From these two different types of experiments, it is clear that the antihypertensive and antianginal effects of bunitrolol are mainly due to its beta-blocking actions, with the alpha 1-blocking action of this drug playing a minor role.     

Autoradiographic analysis of the distribution of beta-adrenoceptors in the dog splenic vasculature.

The technique of in vitro labelling and autoradiography has been used to localize beta-adrenoceptors in sections of the splenic vascular bundle of the dog. Binding of (-)-[125I]-cyanopindolol (Cyp) to sections of splenic vascular bundle equilibrated within 150 min and slowly dissociated after addition of (-)-propranolol. The process was saturable with a dissociation constant (KD) of 40.3 +/- 4.4 pM and Bmax of 18.9 +/- 1.7 fM (in 6 sections). Binding to sections was stereoselective, the (-)-isomer of propranolol being 90 times more effective than the (+)-isomer in competing for (-)-[125I]-Cyp binding. Delineation of beta-adrenoceptor subtypes using the selective antagonists betaxolol (beta 1) and ICI 118,551 (beta 2) indicated that the receptors present were almost exclusively of the beta 2-subtype. Autoradiographic studies under the conditions evaluated in the biochemical experiments showed that beta-adrenoceptors are unevenly distributed in the dog splenic vein, artery and associated nerve bundles. High concentrations of receptors are associated with the splenic nerves and lower but still significant concentrations in the vasculature. Higher resolution studies with nuclear emulsion coated coverslips revealed concentrations of beta-adrenoceptors over cells adjacent to the lumen in veins. In arteries most beta-adrenoceptors were found associated with the medial layer with fewer receptors towards the intima or adventitia. Serial sections of either artery or vein incubated with (-)-[125I]-Cyp in the presence of (-)-propranolol showed low levels of non-localized binding.     

Characterization of the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof

In the present study we investigated the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof. The O-methylation of 2-hydroxyoestradiol (2OHE2) and 2-hydroxyoestriol (2OHE3) displayed saturation kinetics in the intact tissue. The apparent Km and Vmax values for the O-methylation of 2OHE2 were determined to be 0.91 mumol/l and 104 pmol g-1 min-1, respectively, when 2OHE2 was used as substrate; and 1.14 mumol/l and 188 pmol g-1 min-1 when 2OHE3 was used as substrate. The inhibitors of the extraneuronal uptake process (viz; phenoxybenzamine 33 mumol/l; normetanephrine, 46 mumol/l; and deoxycorticosterone acetate 27 mumol/l) failed to inhibit the O-methylation of either 2OHE2 (3.4 mumol/l) or 2OHE3 (3.4 mumol/l) in intact segments of the rabbit thoracic aorta. (-)-Isoprenaline (40 mumol/l) abolished the O-methylation of 2OHE2 (3.4 mumol/l) and markedly reduced that of 2OHE3 (3.4 mumol/l). Pretreatment of tissues with phenoxybenzamine (33 mumol/l) partially restored the O-methylation of 2OHE2 and 2OHE3 in the presence of (-)-isoprenaline (40 mumol/l). The O-methylation of 2OHE2 (5 mumol/l) was significantly reduced in segments of aorta in which the endothelium was removed. The latter reduction could not be attributed to damage to components of the vessel media. The O-methylation of 2OHE2 and (-)-isoprenaline by subcellular fractions of the rabbit aorta also was examined. Both the microsomal and cytosolic fractions were shown to O-methylate 2OHE2 and (-)-isoprenaline, providing evidence for the existence of membrane-bound and soluble forms of COMT in the rabbit aorta. The O-methylation of 2OHE2 by cytosolic and microsomal fractions of the aorta was determined and compared to that of (-)-isoprenaline. The kinetic constants for the O-methylation of 2OHE2 by cytosolic (Km: 0.27 mumol/l; V max: 112 pmol g-1 min-1) and microsomal (Km: 0.15 mumol/l; Vmax: 161 pmol g-1 min-1) fractions were similar. In contrast, the kinetic constants for the O-methylation of isoprenaline by cytosolic (Km: 121 mumol/l; Vmax: 174 pmol g-1 min-1) and membranal (Km: 0.91 mumol/l; Vmax: 105 pmol g-1 min-1) fractions were very different. It is concluded that catechol oestrogens are excellent substrates for catechol-O-methyltransferase (COMT) in the rabbit aorta. Their O-methylation can occur in endothelial structures as well as in the smooth muscle-containing medial sections of the vessel.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of cryptosin with mammalian cardiac beta-adrenoceptors

Cryptosin - a new cardenolide from the leaves of Cryptolepis buchanani R & S was found to be a potent positive inotropic agent. In experiments with dog heart ex vivo, the rise in the cardiac rate associated with an increase in dP/dtmax and left ventricular pressure (LVP) correlated with changes in the beta-adrenoceptor densities as measured by the binding of 3H-Dihydroalprenolol (DHA). A significant change in the beta-adrenoceptor densities was observed when cryptosin was incubated with guinea pig and dog heart sarcolemmal membranes in vitro. Analysis of the binding of 3H-DHA in post-cryptosin treated membranes indicated a non-specific type of interaction of cryptosin with mammalian cardiac beta-adrenoceptors.     

AUTORADIOGRAPHIC ANALYSIS OF (-)-[125I]-CYP BINDING IN MOUSE KIDNEY

The distribution and binding characteristics of the radioligand (-)-[125I]-cyanopindolol (CYP) have been examined in slide mounted mouse kidney sections, using the technique of in vitro labelling and autoradiography. (-)-[125I]-CYP binding to sections was of high affinity (KD = 55.8 pmol/l, s.e.m. = 8.1, n = 4) to a single population of non-interacting sites (nH = 0.95, s.e.m. = 0.01, Bmax = 0.74 fmol/section, s.e.m. = 0.12, n = 4) and stereoselective with respect to the (-)- and (+)-isomers of both propranolol and pindolol. Autoradiographic studies showed that (-)-[125I]-CYP binding was localized to areas in the renal cortex and medulla. Both cortical and medullary binding were abolished by the inclusion of (-)-propranolol (1 mumol/l) in the incubation medium, whereas (-)-isoprenaline (200 mumol/l) selectively abolished cortical binding. Medullary binding could be prevented by the inclusion of the lipophilic compounds cinanserin (10 mumol/l), haloperidol (10 mumol/l) or phentolamine (10 mumol/l), either alone or together or by washing at 37 degrees C. These results suggest that medullary binding sites are lipid rather than receptor-related. In conclusion, in mouse kidney sections, (-)-[125I]-CYP binds to discrete areas in the cortex and medulla. Cortical binding sites have the molecular characteristics of beta-adrenoceptors while medullary binding sites are lipid-related. Caution should therefore be exercised when defining non-specific binding of lipophilic radioligands. The autoradiographic technique is useful for discriminating between receptor and non-receptor binding sites.     

Concomitant glucocorticoid treatment prevents the development of beta-adrenoceptor desensitization in the guinea pig lung

The aim of the present study was to investigate, whether concomitant administration of the synthetic glucocorticoid betamethasone (BM), theophylline (THEO), or the muscarinic antagonist ipratropium bromide (IPRA) could influence the desensitization-associated decrease of beta-adrenoceptors in the guinea pig lung during prolonged in vivo treatment with the beta 2-agonist terbutaline (TER). The animals were sacrificed 20 hrs after the last drug dosage and the lung membrane homogenates were prepared for 3H-dihydroalprenolol (3H-DHA) binding in vitro. Treatment with TER 200 micrograms/kg subcutaneously twice a day for five days decreased by 22% the maximum number of binding sites (Bmax) at saturation in comparison with the saline-treated controls. Concomitant administration of BM 2 mg/kg intraperitoneally abolished this effect of TER, whereas THEO 20 mg/kg or IPRA 5 micrograms/kg failed to modify it. None of the in vivo treatments affected the binding affinity of 3H-DHA. In vitro, TER inhibited in a concentration-dependent manner 3H-DHA binding to the lung membranes of untreated guinea pigs. At high concentrations IPRA, but not THEO or BM, showed some binding to the beta-receptors as well. Thus, it is concluded that glucocorticoids may prevent beta-adrenoceptor desensitization in the lungs via an indirect mechanism, e.g. inhibition of phospholipase A2 enzyme.     

CHARACTERIZATION AND LOCALIZATION OF (—-)[125I]-CYANOPINDOLOL BINDING TO NON-β-ADRENOCEPTOR SITES IN DOG KIDNEY

1. (-)[125I]-Cyanopindolol (CYP) binding to non-beta-adrenoceptor sites in dog kidney was characterized in homogenate preparations and their distribution in sections determined using autoradiography. 2. In homogenate studies, (-)[125I]-CYP bound to a single population of non-interacting sites (Bmax = 5.45, s.e.m. = 1.00 fmol/mg wet weight; nH = 0.99, s.e.m. = 0.01) with high affinity (KD = 3.84, s.e.m. = 0.76 nmol/l, n = 40. 3. In competition studies, compounds selective for alpha- and beta-adrenoceptors, muscarinic cholinoceptors and receptors for 5-HT, histamine and benzodiazepines, calcium channel antagonists, catecholamine uptake inhibitors, MAO inhibitors and adrenergic neurone blockers were ineffective at concentrations of 10 mumol/l. 4. Compounds selective for dopamine D1-receptors (fluphenazine, SCH 23390 and SK & F 82526) and D2-receptors (pimozide, domperidone, spiperone, haloperidol, sulpiride, cis- and trans-flupenthixol) competed with similar affinities (5-25 mumol/l) for (-)[125I]-CYP binding. 5. In autoradiographic studies, (-)[125I]-CYP binding to non-beta-adrenoceptor sites was localized over glomeruli, juxtaglomerular apparatus, distal tubules, blood vessels and medullary rays and tubules. 6. It is concluded that in dog kidney, (-)[125I]-CYP binds to a site closely associated with dopamine receptors.     

Binding characteristics of 3H-dihydroalprenolol to beta-adrenoceptors of rat heart treated with neuraminidase

Binding characteristics of the beta-blockers and beta-agonists with the beta-adrenoceptors were investigated in 3H-dihydroalprenolol (3H-DHA) binding to rat heart membranes treated with neuraminidase. When 60% of the total sialic acid content in the membranes was removed, reproducibility of the binding assay became much better than was attainable without neuraminidase treatment, and the maximum density of beta-adrenoceptors was increased. These data suggest that the binding of the test compounds with the beta-adrenergic receptors in cardiac muscle was under the influence of the sialic acid of the glycocalyx of the membrane. The 3H-DHA binding sites in membranes treated with neuraminidase showed a strict stereo-specificity when tested with propranolol. The ranking order of inhibition of beta-antagonists or agonists is: dl-propranolol greater than oxprenolol greater than alprenolol greater than pindolol greater than YM-09538 greater than labetalol greater than acebutolol greater than atenolol greater than metoprolol greater than sotalol greater than butoxamine greater than practolol as antagonists or l-isoproterenol greater than l-epinephrine greater than l-norepinephrine as agonists. A good correlation (r = 0.91, P less than 0.001) was observed between the Ki values observed by the present binding assay and the pA2 observed in the guinea-pig atria relative to the positive inotropic effect by Bieth et al. (Br. J. Pharmacol. 68, 563-569, 1980), indicating that the present method will be useful for screening new beta-adrenergic receptor antagonists or agonists.     

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.     

血管内皮细胞损伤与动脉硬化的实验研究

目的探讨动脉硬化模型兔腹主动脉管壁厚度与血管内皮细胞损伤及高脂血症的关系。方法采用血管内膜剥脱术在兔腹主动脉上造成特定的损伤,观察高脂餐喂养不同时间各组动物血管狭窄程度和病理改变。结果内膜剥脱3个月和4个月可见兔腹主动脉内膜面不规则增厚,斑块形成。病理检查证实高脂喂养4个月时,动脉的粥样硬化病理改变已属于纤维斑块期。结论高脂血症与血管内皮细胞损伤是引起动脉硬化的危险因素。     

Does chronic prenatal methadone exposure affect beta-receptor subtypes in placental, fetal and maternal brain homogenates?

Computer competition analysis of 3H-DHA (3H-dihydroalprenolol, a nonselective beta-adrenergic radioligand) binding in the presence of unlabeled metoprolol (a beta 1-selective antagonist) indicates the existence of both beta 1- and beta 2-adrenergic receptor subtypes in the rat placenta and confirms previous reports that both beta-adrenoceptors are present in adult rat cortex. In the fetal brain (20th day of gestation), however, only beta 1-receptors were detected. Pregnant rats were chronically exposed to methadone from day 7 to day 20 of gestation via implanted osmotic minipumps (6.3-9.0 mg/kg/day). This treatment schedule did not induce a change in the affinity and density of either beta-receptor subtype in the placental, fetal and maternal brain homogenates. The results are discussed in terms of the reported monoaminergic and opiate receptor functional interactions.     

Diltiazem: lack of myocardial beta-adrenergic receptor-binding capacity

It has been suggested that the mechanism of action of the calcium blocker diltiazem (DZ) is via beta-receptor blockade. In order to test this hypothesis, the effects of DZ on the competitive binding of 3H-dihydroalprenolol (3H-DHA) to myocardial beta-receptors were evaluated and compared to those of a known beta-receptor agonist. Preliminary validation studies indicate that binding sites for 3H-DHA exhibit stereospecificity for isoproterenol (IP) (l-IP>d-IP) and show greater affinity for l-epinephrine compared to l-norepinephrine. In order to test the binding capacity of DZ to beta-adrenergic receptors, binding-concentration relationships were constructed for 3H-DHA (3--60 nM) in the presence of no drugs, l-IP (10(-4) M), or DZ (2.2 x 10(-6) M). 3H-DHA binding was significantly inhibited over the entire concentration range by 1-IP but was unaffected by the other conditions. This study was repeated using two different concentrations of DZ (2.2 x 10(-5) and 2.2 x 10(-7) M) with a similar lack of inhibition of 3H-DHA binding. These data indicate that DZ does not bind to myocardial beta-receptors and, therefore, does not appear to act via a beta-receptor-blocking activity.     

Localization of beta-adrenoceptors in the rabbit ear by light microscopic autoradiography

The beta-adrenoceptor antagonist [125I]cyanopindolol (CYP) was used to localize beta-adrenoceptors in sections of rabbit ear. Biochemical studies demonstrated that the binding was stereoselective, and that the beta-adrenoceptors are predominantly of the beta 2-subtype. Autoradiographic studies using 3H-Ultrofilm or nuclear emulsion coated coverslips showed high concentrations of beta 2-adrenoceptors present in the central ear artery, hyaline cartilage, nerve trunks, epithelium and sebaceous glands.     

Action of the novel selective thromboxane antagonist (3R)-3-(4-fluorophenylsulfonamido)-1,2,3,4-tetrahydro-9-carbazolepropan oic acid on vascular smooth muscle preparations

The action of the thromboxane A2-receptor-antagonist (3R)-3-(4-fluorophenylsulfonamido)-1,2,3,4-tetrahydro-9-carbazo lepropanoic acid (Bay u 3405) on vascular smooth muscle preparations was investigated in vitro. In rabbit aortic rings Bay u 3405 is a potent inhibitor of vasoconstriction produced by thromboxane A2 (TXA2)/PG endoperoxides generated by stimulated human platelets (EC50 1.3 x 10(-6) mol/l), (+/-)-cTA2 (Carbocyclic thromboxane A2 [2 beta(Z),3 alpha-(1E,3R*)-3- (3-hydroxy(1-octenyl)-bicyclo[3.1.1]hept-2-yl-5-heptenoic acid]) (EC50 3.3 x 10(-7) mol/l) and U 46619 (EC50 3.8 x 10(-7) mol/l). In pig circumflex coronary arteries Bay u 3405 was 150 times more potent (EC50 2.6 x 10(-9) mol/l) than in rabbit aorta. In rabbit and rat aorta the concentration-response curves for U 46619 were shifted to the right in a parallel manner and the maximum responses were not suppressed. The Schild-plot yielded a straight line with a slope of 1.14 (rabbit) or 1.29 (rat) and pA2 values of 7.43 and 8.59, respectively. The vasoconstrictive action of other agonists such as KCl, serotonin, histamine, epinephrine, norepinephrine, acetylcholine and angiotensin were not affected. In human platelets inhibition of the TXA2-synthase was seen only at concentrations of Bay u 3405 of 2.4 x 10(-5) mol/l and above. From these findings it is concluded that Bay u 3405 is a potent, competitive TXA2/endoperoxide receptor antagonist in vascular smooth muscle.     

PPARγ激活剂罗格列酮对兔动脉粥样硬化斑块消退的影响

过氧化物酶体增殖激活受体γ(PPARγ)是一个核受体,在血管壁内皮细胞、巨噬细胞/泡沫细胞及血管平滑肌细胞都有较高表达。PPARγ通过对这些细胞的调控作用,还影响着炎性因子的水平,在动脉粥样硬化(AS)疾病中发挥着重要的作用[1]。本实验通过高选择性PPARγ激动剂罗格列酮对高胆     

3H-spiroperidol binding sites in the rabbit superior mesenteric artery. A histoautoradiographic study

The distribution of 3H-spiroperidol binding sites within rabbit superior mesenteric artery was studied in normal as well as 6-hydroxydopamine (6-OHDA) sympathectomized animals using a histoautoradiographic technique. The labelled drug was located in the three layers of the artery (adventitia, media and intima), with the greater density in the media. In the adventitia the radiolabelled drug was located at the level of fibrous and connective cells. In the media 3H-spiroperidol was bound by smooth muscle cells and is found in the cellular membrane of the same cells. 6-OHDA administration causes an increase in the number of 3H-spiroperidol binding sites in the adventitia and as well as a 20-25% increase in the media. The possible existence of a direct dopaminergic innervation of the superior mesenteric artery is discussed.     

Demonstration of α-adrenoceptors in the rabbit heart by [3H]-dihydroergocryptine binding

For direct identification of alpha-adrenoceptors in a membrane fraction of the rabbit heart the potent alpha-adrenoceptor antagonist [3H]-dihydroergocryptine ([3H]-DHE) was used. 1. The binding of [3H]-DHE was saturable with 80 fmol of [3H]-DHE bound/mg protein and of high affinity with an equilibrium dissociation constant (KD) of 11.5 nM. Binding of [3H]-DHE (6 nM) was rapid (t 1/2 = 2 min) and readily reversible. From the ratio of the rate constants for forward (K1 = 1.97 X 10(7) M-1 min-1) and reverse (K2 = 0.206 min-1) reactions a KD-value of 10 nM was calculated, which is in good agreement with that obtained by equilibrium studies. 2. Adrenergic agonists compete for [3H]-DHE binding in an order to potency: (-)adrenaline greater than (-)phenyleprine greater than (-)isoprenaline and adrenergic antagonists in the order: phentolamine greater than yohimbine greater than (-)propranolol. Binding is stereospecific as indicated by the greater potency of (-)adrenaline than (+/-)adrenaline in displacing [3H]-DHE from the binding sites. 3. For comparison the binding of the potent beta-adrenoceptor antagonist (-)[3H]-dihydroalprenolol ((-)[3H]-DHA) was measured in the same membrane fraction. The number and affinity of beta-adrenoceptors amounted to 115 fmol of (-)[3H]-DHA bound/mg protein at saturation and KD = 7.9 nM. Adrenergic agonists compete for (-)[3H]-DHA binding in an order of potency: (-)isoprenaline greater than (-)adrenaline greater than (-)phenylephrine; and adrenergic antagonists in the order: (-)prapranolol greater than phentolamine. 4. It is concluded that in a membrane fraction of the rabbit heart there exist binding sites for [3H]-DHE which have characteristics indistinguishable from alpha-adrenoceptors. Thus the present results are in agreement with previously reported data on the existence of cardiac alpha-adrenoceptors in the rabbit heart (Schümann et al., 1974; Endoh et al., 1976b).     

High affinity non-beta-adrenoceptor binding of beta-adrenergic ligands

Two types of saturable binding, besides the well-known non-specific binding, were found when hydrophobic ligands were used for investigating the vesiculization of beta-adrenoceptors on cultured HeLa and Chang liver cells. The first (compartment I) representing beta-adrenoceptors with high affinity ([3H]DHA 0.8 nM, 125I-CYP 27 pM) and low capacity (10-20 fmol/mg protein), the second (compartment II) had a rather high affinity ([3H]DHA 400 nM, 125I-CYP 30 nM) and a very high capacity (20 000-25 000 fmol/mg protein). The affinity of adrenergic agents for compartment II correlates very well (r = 0.9418) with the calculated hydrophobicity. It is concluded that these types of binding sites might interfere with the determination of adrenoceptor binding sites when hydrophobic ligands are used. When using hydrophobic ligands like these special care should be taken to avoid such interference.