Thermodynamic Study of Iodocyanopindolol β-Adrenoceptors Interactions with Rat Lung and Cerebral Cortex

Abstract

(±)¹²⁵ I-cyanopindolol (±) I CYP) was used to characterize β-adrenoceptors on rat lung and cerebral cortex membranes. The affinity of (±) ICYP was higher for lung (K^d = 64.3 pM) at 37°C. The association reaction of (±) ICYP was faster with lung (k₊₁ = 1.52 × 10⁹ M^?1.min^?1) than with cerebral cortex β-adrenoceptors (k₊₁ = 1.75 × 10⁸ M^?1.min^?1). In both tissues, the dissociation reaction followed a biphasic process with a fast (t ½ = 15.4 min and 5.6 min for lung and cerebral cortex respectively) and a slow component (t ½ = 474 min and 255 min for lung and cerebral cortex respectively). The thermodynamic parameters for (±) ICYP - β-adrenoceptors binding have been determined from kinetics and equilibrium studies, for the two tissues, at several temperatures between 0° and 44° C. For lung and cerebral cortex, Arrhenius plots were linear with different energies of activation. Van't Hoff plot was not linear for lung and the standard enthalpy and entropy changes of (±) ICYP - β-adrenoceptors interaction decreased linearly with temperature : the binding occured with a negative heat capacity change (ΔCp° = -368.9 cal. moles^?1. K^?1) at 25° C. Thermodynamic and kinetic results show that binding of (±) ICYP to lung β-adrenoceptors could involve two successive equilibria with a conformational change of the β-adrenergic receptor.     

Labelling of Rat Brain ß-Adrenoceptors: (3H)CGP-12177 or (125I)Iodocyanopindolol?

Abstract

Binding of (¹²⁵I) iodocyanopindolol (ICYP) and (³H) CGP-12177 to rat brain homogenates was characterized and compared. ICYP was shown to bind to both ß-adrenergic and serotonin1B (5HT_1B) receptors whereas (³H)CGP-12177 only labelled the first ones. The addition of 10 μM serotonin (5HT) prevented ICYP binding to 5HT receptors and under these experimental conditions both ligands labelled a similar total number of ß-adrenoceptors in the different rat brain regions. ICYP displayed a higher affinity for cerebellar (mainly ß₂-subtype) than for cerebral cortex ß-adrenoceptors (mainly ß-subtype) suggesting a subtype selectivity. A multiple displacement binding approach using CGP-20712A, a ß₁-subtype ligand, as competitor revealed a 2.6 fold selectivity of ICYP for the ß₂-adrenoceptor subtype. On the other hand, (³H)CGP-12177 binds only to ß-adrenoceptors and is not subtype selective in the rat brain homogenate. Considering both its high specificity and its lack of subtype selectivity (³H)CGP-12177 seems to be a more suitable ligand than ICYP to non-selectively label ß-adrenoceptors in rat brain.     

Kinetic and Equilibrium Studies of (-)125Iodocyanopindolol Binding to β-Adrenoceptors on Human Lymphocytes: Evidence for the Existence of two Classes of Binding Sites

Abstract

Saturation experiments were performed on intact human peripheral mononuclear leucocytes (MNL) and MNL membranes with (-)¹²⁵Iodocyanopindolol (¹²⁵ICYP) over a large concentration range (1.5-600pmol/l). The corresponding Scatchard plots were curvilinear suggesting two saturable classes of binding sites: A high affinity binding site (B_max1=1000±400 sites/cell, K_d1= 2.1±0.9 pmol/l for intact MNL and B_max1=550±190 sites/cell, K_d1=4.1±0.9 pmol/l for MNL membranes)and a low affinity binding site (B_max2=9150±3590 binding sites/cell, K_d2=440±50 pmol/l for intact MNL and B_max2=11560±4690 sites/cell, K_d2=410±70 pmol/l for MNL membranes). Dissociation of (-)¹²⁵ICYP from MNL was biphasic consisting of a slow dissociating component (dissociation rate constant k_-1=(0.5±0.2)x10^?3 min^?1 for intact MNL and k_-1=(1.0±0.1)x10^?3min^?1 for MNL membranes) and a fast dissociating component (k_-2=(80±20)x10^?3min^?1 for intact MNL and k_-2=(60±10)x10^?3min^?1 for MNL membranes). In dissociation experiments started after equilibration with various (-)¹²⁵ICYP concentrations k_-1 and k_-2 were independent of the equilibrium concentration, whereas the percentual occupancy of the slow and the fast dissociating component varied and was similar to the estimated fractional occupancy of either binding site at the same (-)¹²⁵ICYP concentrations in saturation experiments. The association rate constant was in the same order of magnitude for both binding sites. These results suggest two independent classes of binding sites for (-)¹²⁵ICYP on MNL.     

Effect of Age on the Kinetics of the Binding of Iodocyanopindolol to a Membrane Preparation of Rat Lungs

Abstract

The association (k₊₁) and dissociation (k_-1) rate constants, and the equilibrium thermodynamic binding parameters (ΔG°, ΔH° and ΔS°) of the β-adrenergic ligand [¹²⁵Iodo]cyanopindolol (ICYP) were studied in a crude lung membrane preparation of rats of different ages. There was no difference in k₊₁-values for the different age groups, while the k_-1-values were in all cases difficult to measure: almost no dissociation of ICYP from its binding site occurs. The thermodynamic properties were not affected by age. It is concluded that, in these experimental conditions, age has no effect on the kinetic parameters of the binding of ICYP to the β-adrenoceptors in rat lung.     

Differential Regulation of Transferrin Receptor Gene Expression in Human Hemopoietic Cells: Molecular and Cellular Aspects

Abstract

As we have shown earlier (-)¹²⁵lodocyanopindolol (¹²⁵ICYP) binding to β-adrenoceptors (β-AR) in human mononuclear leucocytes (MNL) yields evidence for the existence of high affinity (B_hiaff) and low affinity (B_loaff) binding sites. We studied the regulation of these 2 classes of binding sites during 240 min of (-)-epinephrine (EPI) infusion (0.1 μg/kg/min) (n=8) in male healthy volunteers. Saturation experiments were performed on MNL membranes with ¹²⁵ICYP over a large concentration range (1–550 pmol/l). Binding parameters were calculated by computer analysis assuming 2 classes of binding sites. We found a preinfusion value of 830±50 [sites/cell] (K_D=1.5±0.2 pmol/l) of B_hiaff binding sites and 5210±510 [sites/cell] (K_D=420±80 pmol/l) of B_loaff. During EPI infusion we observed biphasic modulation of the B_hiaff and an inverse modulation of the B_loaff. After 40 min of EPI B_hiaff increased to 1970±280 [sites/cell] (K_D=4.2±0.8 pmol/l), whereas B_loaff decreased to 2720±280 [sites/cell] (K_D=140±70 pmol/l); despite constant plasma epinephrine concentration (PEC) after 240 min of EPI B_hiaff changed to 1310±240 [sites/cell] (K_D=2.8±1.0 pmol/l) vs. 4370±760 [sites/cell] (K_D=190±100 pmol/l) B_loaff. These results suggest an interdependent inverse modulation of the 2 classes of binding sites for ¹²⁵ICYP on MNL during EPI infusion.     

Pharmacological evaluation of ocular β-adrenoceptors in rabbit by tissue segment binding method

AimsThis study evaluates ocular (iris, ciliary body and ciliary process) and nonocular (atria and lung) β-adrenoceptors in rabbit to characterize the plasma membrane β-adrenoceptors and binding affinities of β-adrenoceptor antagonists.Main methodsThe tissue segment binding method with a hydrophilic radioligand (?)-4-[3-t-butylamino-2-hydroxypropoxy]-[5,7-³H]benzimidazol-2-one ([³H]-CGP12177) was employed.Key findingsSpecific and saturable binding of [³H]-CGP12177 to intact tissue segments was detected by using (±)-propranolol to define nonspecific binding, showing a single population of plasma membrane binding sites with high affinity. Competition experiments with selective β₁- and β₂-adrenoceptor antagonists revealed a single population of β₂-adrenoceptors in ocular tissues and of β₁-adrenoceptors in atria, but mixed populations of β₁- and β₂-adrenoceptors in 70% and 30%, respectively, in lung. A competition curve for timolol was biphasic in lung and its binding affinity for β₂-adrenoceptors was approximately 158-fold higher than for β₁-adrenoceptors, indicating the β₂-selectivity of timolol. In contrast, competition curves for stereoisomers of befunolol, carteolol, and propranolol were monophasic in all tissues. The (?)-enantiomers of these antagonists were more potent than corresponding (+)-enantiomers in displacing from [³H]-CGP12177 binding, and the isomeric potency ratios of befunolol and carteolol were less than those of propranolol.SignificanceThis study with tissue segment binding method suggests that the binding affinity of (?)-enantiomers of β-adrenoceptor antagonists for plasma membrane β-adrenoceptors (β₁-adrenoceptors of atria, β₂-adrenoceptors of ocular tissues, and mixed β₁-/β₂-adrenoceptors of lung) is higher than that of corresponding (+)-enantiomers and their stereoselectivity is different between β-adrenoceptor antagonists.     

Characterization of Rat Cerebral Cortical Beta Adrenoceptor Subtypes Using (-)-[125I]-Iodocyanopindolol

Abstract

(-)-[¹²⁵I]-Iodocyanopindolol ((-)ICYP), used to characterize beta adrenoceptors on membrane preparations from rat cerebral cortex, was shown to have affinity for both beta adrenoceptors and serotonin receptors. Therefore, 10 µM serotonin was added to the assays to prevent (-)ICYP binding to serotonin receptors. Under these conditions, (-)ICYP binding to the cortical membrane preparation was reversible and saturable, and the association reaction was very slow. The dissociation reaction was also very slow, and revealed two affinity states corresponding to a high and a low affinity state. Scatchard analysis showed a single class of binding sites with an equilibrium dissociation constant (K_D) of 20.7 pM, and a maximal density of binding sites (B_max) of 95.1 fmol/mg membrane protein. Displacement binding analyses revealed a potency series of (-) isoproterenol greater than (-) epinephrine equal to (-) norepinephrine, suggesting a predominance of the beta₁ adrenoceptor subtype. Detailed competition ligand binding studies with the selective beta₁ adrenoceptor antagonist ICI-89406 and the selective beta₂ adrenoceptor antagonist ICI-118551, showed that about 70% of the beta adrenoceptor population in the rat cortex is of the beta₁ subtype with the remainder being of the beta₂ subtype.

We conclude that since (-)ICYP binds to both beta adrenoceptors and serotonin receptors, it is important to prevent the binding of (-)ICYP to serotonin receptors by adding a suppressing ligand like excess cold serotonin when assaying beta adrenoceptors. We have presented the first such characterization of rat cerebral cortical beta adrenoceptors with (-)ICYP in this study.     

The beta-adrenergic receptor in human lymphocytes: subclassification by the use of a new radio-ligand, (+/-)-125 Iodocyanopindolol

(±)?¹²⁵Iodocyanopindolol (ICYP), a new radio-ligand with high affinity and specificity to β-adrenoceptors was used to identify and characterized β-adrenergic receptors in human lymphocytes. Binding of ICYP was saturable with 1.56 ± 0.2 fmol ICYP specifically bound/10⁶ cells at maximal occupancy of the sites and of high affinity (K_D=57 ± 7.1pM, N=4. In contrast to ¹²⁵Iodohydroxybenzylpindolol ICYP-binding was not affected by phentolamine (up to 10^?4M) or serotin (up to 10^?5M). Analysis of inhibition of ICYP-binding via a pseudo-Scatchard-plot (“Hofstee-plot”) by β_1-selective (practocol, metaprolol) and β_2-selective (IPS 339, zinterol) adrenergic drugs resulted in linear plots suggesting the existence of a homogeneous population of β-adrenergic receptorsin human lymphocytes. From the resulting K_D-values for practolol (16.8 μM), metoprolol (4.11 μM), zinterol (0.08 μM) and IPS 339 (0.002 μM) is concluded that the β-adrenergic receptor present in human lymphocytes is of the β₂-subtype. According to its low non-specific binding and its high specificity to β-adrenergic receptors ICYP appears to be an ideal ligand for long-term studies on the regulation of β-adrenergic receptors of human lymphocytes.     

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that (±)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist $\text{(±)-[}{}^3\text{H]hydroxybenzylisoproterenol}$ to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k₁=1.37·10⁷·M^?1·min^?1). Dissociation of specific binding by 0.5 mM (?)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k₂ of 0.106·min^?1 and 0.011·min^?1.[³H]Hydroxybenzylisoproterenol binding was saturable (B_max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [³H]hydroxybenzylisoproterenol binding sites, one having high affinity (K_D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity (K_D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [³H]hydroxybenzylisoproterenol binding with the following order of potency=(?)-propranolol>(?)-isoproterenol>(?)-norepinephrine≈ (?)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β₁-adrenergic receptors. Competition curves of [³H]hydroxybenzylisoproterenol binding by the β-agonist (?)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (?)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [³H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (?)-isoproterenol, then number of [³H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (?)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (?)-isoproterenol to displace [³H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [³H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [³H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [³H]dihydroalprenolol in these cells.     

The effects of (±)-, (+)-, and (−)-atenolol,sotalol, and amosulalol on the rat left atria and portal vein

The effects of (±)-, (+)-, and (?)-atenolol, sotalol, and amosulalol alone on the rat left atria and portal vein and on the respective β₁- and β₂-adrenoceptor-mediated responses to isoprenaline have been determined. (±)-Atenolol at 10^?6 M had no effect whereas high concentrations of (+)- and (?)-sotalol, 10^?5–10^?4 M, and (±)-, (+)-, and (?)-amosulalol depressed the response of the rat left atria to cardiac stimulation which indicates membrane stabilizing activity. None of the drugs tested had any effect alone on the rat portal vein. The order of potency as antagonists was (±)-amosulalol > (±)-atenolol > (±)-sotalol at β₁-adrenoceptors and (±)-amosulalol > (±)-sotalol > (±)-atenolol at β₂-adrenoceptors. (±)-Atenolol and (±)-amosulalol are β₁-selective whereas (±)-sotalol is β₂-selective. For each of the racemic β-blockers, the β₁- and β₂-adrenoceptor blocking activity was predominantly due to the (?)-enantiomer. © 1993 Wiley-Liss, Inc.     

Demonstration of alpha 1-adrenoceptors in guinea pig lung using 3H-prazosin.

³H-prazosin, a new radioligand of high specific radioactivity (33 Ci/mmol) was used to characterise postsynaptic (α₁) adrenoceptors in guinea pig lung membranes. Binding was saturable and of high affinity (K_D 0.24 nM) with a Bmax of 54 fmol/mg protein. Adrenergic agonists competed for binding in the order (?)-epinephrine > (?)-norepinephrine ? (?)-phenyl-ephrine > (?)-isoproterenol. (+)-norepinephrine was 100x less potent than (?)-norepinephrine. α-Adrenergic antagonists competed in the order prazosin > WB 4101 > indoramin > phentolamine > haloperidol > chlorpromazine ? piperoxan > yohimbine, indicating that ³H-prazosin binding is probably to α₁-adrenoceptors. Propranolol, methysergide and sulpiride inhibited binding only at high concentrations. Binding of (?)-³H-dihydroalprenolol under identical experimental conditions gave a K_D of 0.93 nM and a Bmax of 870 fmol/mg protein, giving a ratio of beta : α-adrenoceptor binding sites of 16 : 1 in this lung membrane preparation. ³H-prazosin appears to be a useful ligand in studying α₁-adrenoceptors.     

In vitro characterization of skeletal muscle β-adrenergic receptors coupled to adenylate cyclase

[³H]Dihydroalprenolol, a potent ß-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ß-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed [³H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [³H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [³H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions.The binding of [³H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K₁) and dissociation (K₂) rate constants of 2.21 · M^? · min^?1 and 3.21 · 10^?1, respectively, were obtained. The dissociation constant (K_d) of 15 nM for [³H]dihydroalprenolol obtained from saturation binding data closely agreed with the (K_d) derived from the ratio of dissociation and association rate constants (K₂/K₁).Several β-adrenergic agents known to be active on intact skeletal muscle also competed for [³H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [³H]dihydroalprenolol binding sites with a potency of isoproterenol > epinephrine > norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (?)-isomers being more than potent than (+)-isomers. Phenylephrine, an α-adrenergic agonist, showed no effect either on [³H]dihydroalprenolol binding or on adenylate cyclase. Known ß-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [³H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The (K_i) values for the antagonists determined from inhibition of [³H]dihydroalprenolol binding agreed closely with the (K_i) values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [³H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the ß-adrenergic receptor.     

Comparative characteristics of binding kinetics of specific antagonists by α<Subscript>1</Subscript>- and α<Subscript>2</Subscript>-adrenoceptors in rat cerebral cortex membranes

The binding of specific nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosine and [³H]RX821002 has been studied on rat cerebral cortex synaptosomal membranes. It is shown that for α₁-adrenoceptors the ligand-receptor interaction corresponds to the model assuming the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of [³H]prazosine binding to α₁-adrenoceptors were: K _d= 1.56 ± 0.17 nM, B _max = 30.25 ± 1.78 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.94 ± 0.08 nM, B _max = 12.77 ± 3.17 fmol/mg protein, n = 2. For α₂ -adrenoceptors the ligand-receptor interaction corresponded to the same model. For α₁ - and α₂-adrenoceptor antagonists the dissociation constants (K _d) are approximately equal (1.56 ± 0.17 and 1.94 ± 0.08 nM, respectively), but the concentration of α₂-adrenoceptors is two times lower than that of α₁-adrenoceptors ( 12.77 ± 3.17 and 30.25 ± 1.78 fmol/mg protein, respectively). The efficiency (E = B _max/2K _d) of the ligand binding to α₁-adrenoceptors is 2.3 times higher than that to α₂-adrenoceptors (7.46 ± 1.32 and 3.29 ± 0.68 fmol/mg protein/nM, respectively. The data suggest that α₁- and α₂ -adrenoceptors in rat cerebral cortex exist as dimers.     

High affinity beta-2-adrenergic receptors in mononuclear leucocytes: similar density in young and old normal subjects

In normal subjects beta-adrenergic responsiveness in the cardiovascular system has been shown to be impaired with increasing age. In order to correlate reduced hormonal responsiveness to an age-related defect at the receptor level, high affinity beta-adrenergic receptors in homogenates of human mononuclear leucocytes have been studied with a (?)-³H-dihydroalprenolol (³H-DHA) binding assay. The binding sites have been characterized by rapid kinetics, saturability, structural and sterospecificity. Binding equilibrium was obtained within 16 minutes at 37° and was reversed by 50% within 10.6 minutes. In 22 healthy subjects a binding capacity of 60 ± 8 fmol/mg protein and an equilibrium dissociation constant (K_D) of 0.6 ± 0.05 nM was found. Beta-adrenergic agonists displaced ³H-DHA binding with a potency order of isoproterenol > adrenaline > noradrenaline. The (?) isomers of beta-adrenergic agonists and antagonists were one to two orders of magnitude more potent as inhibitors of ³H-DHA binding than their corresponding (+) isomers. The binding capacity and affinity of the beta-adrenergic receptors did not differ in old, as compared to young normal subjects. Leucocytes from 14 individuals 18–40 years old had an average density of 53 ± 4 fmol/mg protein, while the average density in leucocytes from 8 individuals aged 53–65 years was 67 ± 8 fmol/mg protein. The K_D was 0.6 ± 0.05 nM in both groups. In conclusion, an age-related decrease of beta-adrenergic receptor-mediated cardiovascular functions does not seem to be reflected in the properties of beta-adrenergic receptors of mononuclear leucocytes.     

Serotoninergic agonists and antagonists are the modulators of α1-Adrenoceptor activity in rat brain cortical membranes

The effects of activation and inhibition of serotonin receptors by serotonin (5-HT) and mianserin on the specific nonselective α₁-antagonist [³H]prazosine binding in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction of α₁-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and the binding of two ligand molecules to the receptor. The parameters of [³H]prazosine binding to α₁-adrenoceptors were as follows: K _d =1.85 ± 0.16 nM, B _max = 31.1 ± 0.3 fmol/mg protein, n = 2. In case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K _d1 = 0.61 ± 0.04, K _d2 = 3.82 ± 0.15 nM, B _m1 = 6.6 ± 0.7, B _m2 = 25.6 ± 0.4 fmol/mg protein, n = 2. The sensitivity of the high-affinity pool increased threefold and the sensitivity of the low-affinity pool decreased twofold as compared to the control. The value of maximal reaction (B _max) did not change. In the case of inhibition of 5HT-receptors by mianserin, radioactive ligand is bound to α₁-adrenoceptors according to the same model as in the control conditions. The affinity of α₁-adrenoceptors to [³H]prazosine decreases twofold and the concentration increases (K _d = 3.97 ± 0.12 nM, B _max = 40.0 ± 0.5 fmol/mg protein). The data suggest that α₁-adrenoceptors in rat cerebral cortex exist as a dimer. The modulatory effects of serotonin and mianserin on the specific binding of [³H]prazosine to α₁-adrenoceptors was detected, manifesting itself as changes in the binding parameters and in the general character of ligand-receptor interactions.     

Modulation of alfa-adrenoceptor activity by beta-adrenoceptor agonists and antagonists in rat brain cortex membranes

The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosin and [³H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α₁- and α₂-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [³H]prazosin binding to α₁-adrenoceptors were: K _d = 1.85 ± 0.16 nM, B _max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.57 ± 0.27 nM, B _max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α₁- and α₂-adrenoceptors occurred according to the same model. The affinity to [³H]prazosin and the concentration of active α₁-adrenoceptors increased by 27% (K _d = 1.36 ± 0.03 nM) and 84% (B _max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α₂-adrenoceptors to [³H]RX821002 decreased by 56% (K _d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [³H]prazosin and [³H]RX821002, two pools of receptors were detected with the following parameters: K _d1 = 1.13 ± 0.09, K _d2 = 6.07 ± 1.06 nM, B _m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K _d1 = 0.61 ± 0.02, K _d2 = 3.41 ± 0.13 nM, B _m1 = 1.88 ± 0.028, B _m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B _max) increased twofold for both ligands. It was suggested that α₁- and α₂-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α₁- and α₂- adrenoceptors was revealed, which was manifested in the activating effect on the [³H]prazosin binding parameters, in the inhibitory effect on the [³H]RX821002 binding parameters, and in a change of the general character of binding for both ligands.     

Presence of D2-dofamine Receptors in Human Term Placenta

Abstract

We studied the binding of [³H]-spiperone on human term placental membranes. This binding reached plateau level after 30 min incubation at 37°C and was reversed (t_1/2 ～ 5 min) by addition of an excess of unlabeled spiperone. Scatchard analysis of saturation experiments with increasing doses of [³H]-spiperone (0–25 nM) showed one class of high affinity binding sites with a dissociation constant (Kd) of 14 ± 2 nM and a maximal binding capacity (Bmax) of 222 ± 9 fmoles/mg protein. The affinity of 5 competitors was determined in competitive binding assays. The D₂-dopamine antagonists were the most potent inhibitors: Ki for spiperone and haloperidol were 8 ± 2 and 56 ± 22 nM respectively. Dopamine inhibited [³H]-spiperone binding with a Ki of 570 ± 50 μM whereas Schering 23390 (D₁ antagonist) and propranolol (β-adrenergic antagonist) were without effect. The binding was also inhibited by 100 μM GTPγS (38 ± 8% inhibition), indicating that the dopamine receptor is coupled with a GTP binding protein. These results demonstrate for the first time the presence of D₂-dopamine receptors in human placenta.     

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process

The interaction between interleukin IL-1α and PGE₂ on P388D₂ on cells has been investigated. Preincubation of murine macrophage-like cells, P388D₁, with IL-1α (0–73 pM) reduced the binding of PGE₂ to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1α decreased the PGE₂ binding by lowering both the high and low affinity receptor binding capacities (from 0.31 ± 0.02 to 0.12 ± 0.01 fmol/10⁶ cells for the high affinity receptor binding sites and from 2.41 ± 0.12 to 1.51 ± 0.21 fmol/10⁶ cells for the low affinity receptor binding sites). However, the dissociation constants of the receptor of the IL-1α-treated cells remained unchanged. Inhibition of PGE₂ binding IL-1α did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1α inhibits the binding of PGE₂ to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE₂.     

3H]epinephrine and [3H]norepinephrine binding to alpha-noradrenergic.

(±)-[³H]Epinephrine and (?)-[³H]norepinephrine bind saturably to calf cerebral cortex membranes under appropriate incubation conditions in a fashion indicating that they label α-noradrenergic receptors. Binding of the two [³H]catecholamines is saturable with dissociation constants of 20–30 nM. Binding is stereoselective with (?)-norepinephrine displaying about twenty times greater affinity than (+)-norepinephrine. The relative potencies of catecholamines in competing for these binding sites parallels their relative pharmacologic effects at α-noradrenergic receptors in numerous tissues. Thus, (?)-epinephrine is 2–3 times more potent than (?)-norepinephrine and 500 times more potent than (?)-isoproterenol. Binding is inhibited by low concentrations of the α-antagonists phentolamine and phenoxybenzamine but not by the β-antagonist propranolol.     

Serotoninergic agonist and antagonist are modulators of the α2-adrenoceptor activity in rat brain cortex membranes

The influence of activation and inhibition of serotonin receptors by serotonin (5HT) and miancerin on binding of specific nonselective α₂-antagonist [³H]RX821002 in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction for α₂-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of the [³H]RX821002 binding to α₂-adrenoceptors were as follows: K _d = 1.57 ± 0.276 nM, B _max = 7.24 ± 1.63 fmol/mg protein, n = 2. In the case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K _d1 = 0.82 ± 0.06; K _d2 = 2.65 ± 0.22 nM; B _m1 = 1.65 ± 0.23; B _m2 = 4.20 ± 0.11 fmol/mg protein; n = 2. The affinity of high-affinity receptors increased twofold and the affininty of low-affinity receptors decreased by 69% as compared to control values. The concentration of high-affinity receptors decreased 4.4-fold, and of low-affinity, 1.7-fold. The value of maximal reaction (B _max) decreased by 20%. In the case of miancerin-induced inhibition of 5HT-receptors the character of ligand binding also changed; two pools of receptors were detected with the following parameters: K _d1 = 0.48 ± 0.09; K _d2 = 3.79 ± 0.71 nM; B ₁ = 0.63 ± 0.17; B ₂ = 4.75 ± 0.21 fmol/mg protein; n = 2. The affinity of high-affinity receptors pool increased by 70% and that of low-affinity receptors decreased by 76% as compared to control values. The concentration of active high-affinity and low-affinity α₂-adrenoceptors decreased by 70% and 141%, respectively. The total amount of the receptors (B _max) decreased by 26%. The data suggest that α₂-adrenoceptors in rat cerebral cortex exist as dimers. Modulatory effects of serotonin and miancerin on specific antagonist binding to α₂-adrenoceptors may be accomplished by altering the character and binding parameters of the nonselective α₂-antagonist [³H]RX821002.