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.     

Decreased response with age of the cardiac catecholamine sensitive adenylate cyclase system

The cardiac β-adrenergic coupled adenylate cyclase system was examined in young and old male Wistar rats. The concentration of binding sites for (?) ³H-DHA in membranes prepared from cardiac ventricles was 21.1 ± 2.78 (SD) fmoles/mg protein in 3–4 month old rats (young rats) and 31.2 ± 2.20 fmoles/mg protein in 24 month old rats (old rats). The dissociation constant, K_D was 4.3 ± 1.8 nM and 6.7 ± 1.7 nM for young and old rats, respectively. Various compounds were used to study the characteristics of activation of adenylate cyclase in homogenates from cardiac ventricles. Basal adenylate cyclase was reduced 30% in old animals compared to young (6.1 pmoles/min/mg protein in 24 month vs. 8.6 pmoles/min/mg protein in 3–4 month). (?)Isoproterenol (10^?5M) alone stimulated adenylate cyclase greater than two-fold in young rats (10.6 pmoles/min/mg protein above basal) and this stimulation was 34% lower in old animals. GppNHp (100 μM), fluoride (10 mM), and forskolin (100 μM) activation of adenylate cyclase above basal was reduced 38, 37, and 34%, respectively, in the old animals. No significant changes between the two groups were noted in the apparent affinity of GppNHp either alone or in the presence of (?)isoproterenol nor in the affinities of catecholamine agonists for activation of cyclase. These results suggest a reduction in the amount of functional regulatory protein or possibly cyclase in 24 month old rat ventricular tissue compared to 3–4 month old tissue. However, this data does not rule out the possibility of altered molecular interactions of a full complement of regulatory protein (s) with β-adrenergic receptor and/or catalytic adenylate cyclase.     

α1- and α2-adrenergic receptors in rat corpus striatum

Rat corpus striatum contained α₂-adrenergic receptor which were labelled with [³H]clonidine (95 ± 6 fmol/mg protein). The affinity of these receptors (K_d = 1.3 to 3.6 nM) was similar to that found in cerebral cortex. Five days after kainic lesions, the number of α₂-adrenergic receptors had dropped by half, suggesting that their location might be neuronal. One month after lesions, the number of α₂-adrenergic receptors had risen to that of the controls and was higher after two months. This increase would suggest a glial localization of the α₂-adrenergic receptor. We have previously described the presence of α₂-adrenergic receptors in primary astrocyte cultures (Ebersolt et al., 1981). Rat corpus striatum contained less α₁-adrenergic receptors than α₂-adrenergic receptors. They were labelled with [³H]prazosin (28 ± 1.9 fmol/mg protein) and were only slightly altered 5 days after kainic acid lesions (?20%). In addition to these classical α₁-adrenergic receptors, rat corpus striatum also contained [³H]WB4101 binding sites having high affinity for WB4101 (2–5 nM) and norepinephrine (1 μM) but a very low affinity for prazosin (4.4 μM). The exact nature of these sites remains unknown.     

II. Beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in the developing rat lung

β-Adrenergic receptors were identified in membrane fractions of fetal and postnatal rat lung with the β-adrenergic antagonist (?)?[³H] dihydroalprenolol, (?)?[³H] DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to day 28 of postnatal life, 46±7 to 491±69 femtomole·mg^?1 protein. Neither the K_D, approximately 0.8nM for [³H]DHA, nor the β-adrenergic subtype changed with age. Classical agonists competed for the β-receptor with properties characteristic of β₂-adrenergic binding. Analysis of the inhibition of receptor binding by selective β-adrenergic agents demonstrated approximately 75% β₂ and 25% β₁ β-adrenergic subtypes in fetal rat lung membranes. The increase in β-adrenergic receptor during development was associated with adenylate cyclase activity which was sensitive to catecholamines at all ages studied, supporting the possible role of the β-adrenergic receptor system in the postnatal regulation of pulmonary function.     

Human platelet vasopressin receptors

Specific saturable binding of 125I-arginine-vasopressin to human platelets is described. For ten normal volunteers the mean (+/- S.D.) KD is 5.6 nM (+/- 2.1) and the mean (+/- S.D.) Bmax is 115 fmoles/mg protein (+/- 30). Association studies indicate that equilibrium is reached after 90 minutes on ice. Pharmacological inhibition studies with analogues indicate that the platelet receptor is very similar to the kidney medulla receptor. The function of the receptor may involve serotonin release and platelet aggregation. Vasopressin binding to platelets should provide a readily means of assessing vasopressin receptor function in man.     

Human platelet alpha 2-adrenergic receptors: labeling with 3H-yohimbine, a selective antagonist ligand

³H-Yohimbine, a potent and selective pharmacological antagonist of α₂-adrenergic receptors, labeled human platelet membrane α₂-receptors with high affinity. Binding was rapid and reversible at 25°C. Both saturation and kinetic experiments indicated a single order of binding sites, with an equilibrium K_D value of 1.0–1.5 nM. Low Mg²⁺ concentrations increased the K_D for ³H-yohimbine without altering the B_max. The ³H-yohimbine site exhibited α₂-receptor specificity: (?)-norepinephrine and (?)-isoproterenol were 4.8 and 330 times less potent than (?)-epinephrine; (?)-catecholamines were 17–35 times more potent than corresponding (+)-catecholamines; the selective α₁-antagonist prazosin was 340 times less potent than yohimbine. Catecholamine agonists exhibited shallow curves in inhibiting ³H-yohimbine binding, with pseudo-Hill coefficients (n_H) of less than 1.0, whereas the n_H of antagonists was 1.0. No specific binding of ³H-prazosin to platelet membranes was observed, indicating the absence of α₁-receptors. ³H-Yohimbine labeled fewer platelet sites than did ³H-dihydroergocryptine under identical conditions (80 vs 130 receptors/ cell), and may be a more specific and useful antagonist probe of platelet α₂-receptors than ³H-dihydroergocryptine.     

Endogenous and Exogenous Regulation of Human α- and β-Adrenergic Receptors

Abstract

Regulation of human β2-adrenergic receptors in lymphocytes (determined by (±)-125 iodocyanopindolol (ICYP) binding) and α₂-adrenergic receptors in platelets (determined by ³H-yohimbine binding) was studied. While α₂-adrenergic receptor number did not change with age, a significant negative correlation between the number of α₂-adrenergic receptors and age was found; plasma catecholamines, on the contrary, were elevated in the elderly.In healthy women during normal menstrual cycle the number of α₂-adrenergic receptors decreased with increasing plasma estradiol levels.Incubation of lymphocyte membranes with isoprenaline (100 μM) and of platelet membranes with clonidine (1-100 μM) led to a reduction of the number of β₂- and α₂-receptors, respectively, without changes in the K_D-values. Treatment of hypertensive patients with clonidine (3x150 μg/die) for 7 days reduced the number of α₂-adrenergic receptors in platelets. In platelet membranes from such treated patients inhibition of ³H-yohimbine binding by clonidine and adrenaline was not affected by 10-⁴MGTP. It is concluded, that human α- and β-adrenergic receptors undergo regulatory mechanisms similar to those recently described for adrenergic receptors in a variety of animal models.     

Preponderance of alpha 2- over beta 1-adrenergic receptor sites in human fat cells is not predictive of the lipolytic effect of physiological catecholamines

Adrenergic control of human fat cell lipolysis is mediated by two kinds of receptor sites that are simultaneously stimulated by physiological amines. To establish a correlation between the binding characteristics of the receptor and biological functions, the ability of physiological amines to stimulate or inhibit isolated fat cell lipolysis in vitro was compared to the beta- and alpha 2-adrenoceptor properties of the same fat cell batch. The beta-selective antagonist (-)[3H]dihydroalprenolol ([3H]DHA) and the alpha 2-selective antagonists [3H]yohimbine ([3H]YOH) and [3H]rauwolscine ([3H]RAU) were used to identify and characterize the two receptor sites. Binding of each ligand was rapid, saturable, and specific. The results demonstrate 1) the weaker lipolytic effect of epinephrine compared with norepinephrine. This can be explained by the equipotency of the amines at the beta 1-sites and the higher affinity of epinephrine for alpha 2-adrenergic receptors. 2) The preponderance of alpha 2-adrenergic receptor sites labeled by [3H]YOH (Bmax, 586 +/- 95 fmol/mg protein; KD, 2.7 +/- 0.2 nM) or [3H]RAU (Bmax, 580 +/- 100 fmol/mg protein; KD, 3.7 +/- 0.1 nM). These two ligands can be successfully used to label alpha 2-adrenergic receptor sites. 3) The beta 1-adrenergic receptor population labeled by [3H]DHA(Bmax, 234 +/- 37 fmol/mg protein; KD, 1.8 +/- 0.4 nM), although a third as numerous as the alpha 2-adrenergic population, is responsible for the lipolytic effect of physiological amines and is weakly counteracted by simultaneous alpha 2-adrenergic receptor stimulation under our experimental conditions. It is concluded that, in human fat cells, the characterization of beta 1- and alpha 2-adrenergic receptors by saturation studies or kinetic analysis to determine affinity (KD) and maximal number of binding sites (Bmax) is not sufficient for an accurate characterization of the functional adrenergic receptors involved in the observed biological effect.     

Identification and ontogeny of beta-adrenergic receptors in fetal rabbit lung

High affinity (Kd=0.2 nM), low capacity (48 fmoles per mg protein), stereospecific binding sites, with properties characteristic of the β₁-subtype of β-adrenergic receptors, have been detected in fetal rabbit lung membranes as early as the 22nd day of gestation. The concentration of the receptor did not change significantly between the 22nd and 26th day of gestation, but increased 3-fold between the 26th and 29th day, reaching a level of 198 fmoles per mg protein. A further increase (from 198 to 315 fmoles per mg protein) in receptor concentration was observed in adult female rabbits. The increase in the levels of pulmonary β-adrenergic receptors between the 26th and 29th day of gestation is temporally related to the increase in surfactant release into the alveolar spaces of the fetal lung. Thus β-adrenergic agonists may act directly on the fetal lung to regulate surfactant secretion.     

Characteristics of an Adenylate Cyclase Coupled β-Adrenergic Receptor in a Smooth Muscle Tumor Cell Line

Abstract

We have shown that binding of ³H-dihydroalprenolol ([³H] DHA) to DDT₁ MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [³H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [³H]-DHA binding site of DDT₁ cell membranes was isoproterenol (K_i = 0.20 ± 0.07 μM) > epinephrine (K_i = 0.4 ± 0.2 μM) > norepinephrine (K_i = 66.5 ± 5.15 μM) consistent with a β₂-selective receptor interaction. Zinterol, a β₂-selective antagonist, (K_i = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β₁-selective antagonist (K_i = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT₁ cells possess a pure population of β₂-adrenergic receptors. Finally, we have shown that DDT₁ MF-2 cell β₂-adrenergic receptor is functionally coupled to adenylate cyclase via a G/F protein complex as demonstrated in part by a guanine nucleotide requirement for isoproterenol stimulation of adenylate cyclase activity. In addition, guanine nucleotide mediated a reduction in the affinities of isoproterenol and epinephrine for the [³H]DHA binding site.     

Characterization of α2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[³H]Yohimbine, a potent α₂-adrenergic antagonist, was used to label the α₂-adrenergic receptors in membranes isolated from human platelets. Binding of [³H]yohimbine to platelet membranes appears to have all the characteristics of binding to α₂-adrenergic receptors. Binding reached a steady state in 2–3 min at 37°C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10^?5 M;$\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.37}\text{min}$). [³H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [³H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (?) isomer was 11-times more potent than the (+) isomer. Cathecholamine agonists competed for the occupancy of the [³H]yohimbine-binding sites with an order of potency: clonidine > (?)-epinephrine > (?)-norepinephrine >> (?)-isoproterenol. The potent α₂-adrenergic antagonist, phentolamine, competed for the sites whereas the β-antagonist, (±)-propanolol, was a very weak inhibitor. 0.1 mM GTP reduced the bindng affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonine competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [³H]yohimbine binding. These results suggest tht [³H]yohimbine binding to human platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label α₂-adrenergic receptors.     

Glomerular thromboxane A2/prostaglandin H2 receptors: characterization and effect of adriamycin-induced nephrotic syndrome

We have characterized the thromboxane (TX) A₂/prostaglandin (PG) H₂ receptor in glomeruli isolated from the rat using the agonist radioligand [¹²⁵I]-BPO. Binding of [¹²⁵]-BOP was highly specific, stereoselective, and to a single class of high affinity binding sites (K_d = 1/16 ± 0.22 nM and B_max = 348 ± 32 fol/mg protein; n = 6). Binding of [¹²⁵I]-BOP was competed for by the agonist ONO11113 (K_d = 50.8 ± 8.0 nM; n = 4) and the antagonists SQ29548 (K_d = 15.8 ± 1.0 nM; n = 3), L657925 (K_d = 12.1 ± 2.2 nM; n = 3) and L65796 (K_d = 1642 ± 135 nM; n = 3). I-BOP also produced a TXA₂/PGH₂ receptor-mediated rise in [CA²⁺]_i in isolated glomeruli In adriamycin-induced nephrotic syndrome in the rat, the development of proteinuria is reported to be dependent on increased renal TXA₂ production. We therefore examined whether or not changes in glomerular TXA₂/PGH₂ receptors occur between control and nephrotic rats. No changes in expression of affinity of either glomerular or platelet TXA₂/PGH₂ receptors were observed. K_d and B_max values for isolated isolated glomeruli were 1.45 ± 0.24 nM and 406 ± 72 fmol/gm for controls and 1.22 ± 0.25 nM and 321 ± 62 fmol/gm for nephrotic rats (n = 6).     

Interaction of clonidine and clonidine analogs with human platelet α2-adrenergic receptors

Several new clonidine analogs were synthesized and their ability to inhibit [³H] phentolamine binding to human platelet α₂-adrenergic receptors was tested. The order of potency and calculated dissociation constants for clonidine and its analogs were as follows: clonidine (0.020 ± 0.005 μM) >p-aminoclonidine (0.100 ± 0.010 μM) > hydroxy-phenacetyl-aminoclonidine (0.20 ± 0.03 μM) >p-dansyl clonidine (1.00 ± 0.20 μM) >t-boc-tyrosine clonidine (1.80 ± 0.60 μM). Thus, p-amino substitution reduces α₂-adrenergic affinity in the platelet system. The effects of clonidine and its p-amino analogs on platelet adenylate cyclase were also evaluated. This enzyme is inhibited by epinephrine acting via α₂-adrenergic receptors. Both clonidine and p-aminoclonidine cause slight inhibition of basal adenylate cyclase and reverse the inhibition induced by epinephrine. These observations indicate that clonidine is a partial agonist for platelet α₂-adrenergic receptors.     

α1-Adrenergic Receptors of A Smooth Muscle Cell Line: Guanine Nucleotides Do Not Regulate Agonist Affinities

Abstract

Using [³H]-dihydroergocryptine, we have identified in membranes prepared from the DDT₁ MF-2 smooth muscle cell line a binding site with characteristics of the α₁-adrenergic receptor. Specific binding (90–95% of total binding) was saturable with a binding site concentration of 197±44 fmol/mg protein and was of high affinity with a dissociation constant of 1.7±0.4 nM. The order of agonist competition for the binding site was epinephrine (K_i=2.3±0.5μM) ≥ norepinephrine (K_i=4.4±1.3μM) ? isoproterenol (K_i=195.5±27.6μM), consistent with an α-adrenergic interaction. Computer modelling of competition curves obtained with prazosin (α₁-selective) and yohimbine (α₂-selective) indicated that the DDT₁ cell αa-adrenergic receptor was predominantly (>95%) of the α₁-subtype. Guanine nucleotides, either GTP or 5'-guanylylimidodiphosphate, did not reduce the affinity of either epinephrine of phenylephrine for the [3H]-dihydroergocryptine binding site.     

The bindings of 3H-prazosin and 3H-yohimbine to alpha adrenoceptors in the guinea-pig stomach

Alpha adrenoceptor subtypes have been investigated by radioligand binding study in guinea-pig stomach using 3H-prazosin and 3H-yohimbine. The specific 3H-prazosin binding to guinea-pig stomach was saturable and of high affinity (KD = 1.4 nM) with a Bmax of 33 fmol/mg protein. Specific 3H-yohimbine binding to the tissue was also saturable and of high affinity (KD = 25.5 nM) with a Bmax of 150 fmol/mg protein. Adrenergic drugs competed for 3H-prazosin binding in order of prazosin greater than phentolamine greater than methoxamine greater than norepinephrine greater than clonidine greater than epinephrine greater than yohimbine. These drugs competed for 3H-yohimbine binding in order of yohimbine greater than phentolamine greater than clonidine greater than epinephrine greater than norepinephrine greater than prazosin greater than greater than prazosin greater than methoxamine. We also examined whether dopamine receptors exist in guinea-pig stomach, using radioligand binding study. Specific binding of 3H-spiperone, 3H-apomorphine, 3H-dopamine and 3H-domperidone was not detectable in the stomach. Dopaminergic drugs such as dopamine, haloperidol, domperidone and sulpiride competed for 3H-prazosin binding in order of haloperidol greater than domperidone greater than dopamine greater than sulpiride. Metoclopramide, sulpiride and dopamine competed for 3H-yohimbine binding in order of metoclopramide greater than sulpiride greater than dopamine. These results suggest that guinea-pig stomach has alpha 1 and alpha 2 adrenoceptors and has no specific dopamine receptors. It is also suggested that some dopamine receptor antagonists such as domperidone, haloperidol, sulpiride and metoclopramide have antagonistic actions on alpha adrenoceptors.     

Demonstration of human platelet β-adrenergic receptors using 125I-labeled cyanopindolol and 125I-labeled hydroxybenzylpindolol

The radioiodinated pindolol analogs ¹²⁵I-labeled cyanopindolol ([¹²⁵I]CYP) and ¹²⁵I-labeled hydroxybenzylpindolol ([¹²⁵I]HBP) have been used to study binding to human platelet β-adrenergic receptors. [¹²⁵I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (K_d) of 14±3 pM and maximal binding capacity (B_max) of 18±4 fmol/mg protein. Binding of [¹²⁵I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8·10⁷ s^?1·M^?1 and 3.8·10^?4 s^?1, respectively. [¹²⁵I]HBP binds to a saturable class of platelet membrane sites with a K_d of 50±10 pM and B_max of 32±6 fmol/mg protein. [¹²⁵I]HBP also binds to a saturable class of sites on intact platelets with a K_d of 58±14 pM and B_max of 24±4 molecules per platelet. Binding of [¹²⁵I]CYP and [¹²⁵I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (?) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol ? isoproterenol > epinephrine > practolol > norepinephrine > phenylephrine. These observations indicate that [¹²⁵I]CYP and [¹²⁵I]HBP bind to platelet sites which have the pharmacological characteristics of β-adrenergic receptors but which are not typical of either the β₁ or β₂ sub-type.     

Interaction of the synthetic peptide octarphin with rat adrenal cortex membranes

The synthetic peptide octarphin (TPLVTLFK, fragment 12?C19 of ??-endorphin), a selective agonist of the nonopioid ??-endorphin receptor, was labeled with tritium yielding specific activity of 28 Ci/mmol. The binding of [³H]octarphin to rat adrenal cortex membranes was studied under normal conditions as well as after cold and heat shocks. It was found that under normal conditions [³H]octarphin specifically binds to the membranes with high affinity: K _d1 = 36.3 ± 2.5 nM, B_max1 = 41.0 ± 3.8 pmol/mg protein. The specific binding of [³H]octarphin to the membranes was inhibited by unlabeled ??-endorphin (K _i = 33.9 ± 3.6 nM) and the agonist of the non-opioid receptor decapeptide immunorphin (K _i = 36.8 ± 3.3 nM). Unlabeled naloxone, [Leu⁵]- and [Met⁵]enkephalins, ??- and ??-endorphins, and corticotropin were inactive (K _i > 1 ??M). Both cold and heat shocks decreased the binding affinity: K _d2 = 55.6 ± 4.2 nM and K _d3 = 122.7 ± 5.6 nM, respectively. In both cases, the maximal binding capacity of the receptor did not change. Thus, even a short-term thermal shock significantly affects the sensitivity of the non-opioid ??-endorphin receptor of adrenal cortex membranes.     

A specific, photolabile and irreversible antagonist (L662,025) of the PAF-receptor

PAF (0.2 microM) induced maximal platelet aggregation in human PRP and [3H]-PAF (1-5 nM) binding to platelet membrane preparations had Kd value of 3.8 nM and Bmax of 200 fmoles/mg of protein. Without UV irradiation, a synthetic azido tetrahydrofuran derivative L662,025 was a reversible and competitive PAF-receptor antagonist with IC50 values of 5.6 +/- 0.3 microM (platelet aggregation) and 1.0 +/- 0.25 microM (receptor binding). Photolysis of L662,025 in the presence of PRP produced an irreversible inhibition of platelet aggregation and specific binding of [3H]-PAF (1 nM). L662,025 did not affect collagen- or ADP-induced human platelet aggregation before or after photolysis. It is a new probe that can be used to identify and characterize the PAF-receptor.     

Kinetics of [3H]prazosin and [3H]dihydroalprenolol binding to α1- and β-adrenoreceptors in rat brain cortex membranes

The binding of nonselective α₁- and β-adrenoreceptor antagonists [³H]prazosin and [³H]dihydroalprenolol ([³H]DHA) to rat cerebral cortex synaptosomal membranes has been studied. It is found that ligand-receptor interactions of α₁-adrenoreceptors fit into a single receptor pool model, which assumes the binding of two ligand molecules to one receptor molecule. The parameters of [³H]prazosin binding to α₁-adrenoreceptors are as follows: K _d = 2.58 ± 0.20 nM; B _m = 2.95 ± 1.12 fmol/mg protein; Hill coefficient, n = 2. For β-adrenoreceptors, ligand-receptor interactions fit into a model assuming the presence of two receptor pools in the same effector system and binding of two ligand molecules to one receptor molecule. The corresponding parameters of the [³H]DHA binding to β-adrenoreceptors are as follows: K _d1 = 0.74 ± 0.09 nM; K _d2 = 7.63 ± 0.70 nM; B _m1 = 25 ± 2 fmol/mg, B _m2 = 48 ± 2 fmol/mg, n ₁ = 2; n ₂ = 2. We suggest that in rat cerebral cortex membranes α-and β-adrenoreceptors exist as dimers.