Allosteric effects of atropine and carbachol on the activity of α<Subscript>2</Subscript>-adrenoceptors in rat brain cortex membranes

Activation and inhibition of muscarinic cholinoceptors by atropine and carbachol are shown to exert allosteric effects on the binding of specific nonselective α₂-adrenoceptor antagonist [³H]RX821002 in rat brain cortex membranes. The ligand-receptor interaction for α₂-adrenoceptors corresponded to the model suggesting the presence of one homogeneous pool of receptors with two specific binding sites. The parameters of the [³H]RX821002 binding were as follows: [³H]RX821002 -K _d = 1.94 ± 0.08 nM, B _max = 13.4 ± 1.8 fmol/mg protein, n = 2. The inhibition of muscarinic cholinoceptors by atropine induced an increase of affinity (K _d = 1.36 ± 0.12 nM) and a decrease of the α2-adrenoceptor density (B _max = 10.18 ± 0.48 fmol/mg protein). The muscarinic cholinoceptor agonist carbachol induced an increase of the affinity (K _d = 1.56 ± 0.05 nM) and quantity of binding sites (B _max = 16.61 ± 0.29 fmol/mg protein). As a result, under the influence of atropine and carbachol, the efficiency of binding (E = B _max/2K _d) increased from 3.50 ± 0.40 to 5.60 ± 0.79 and 6.86 ± 0.20 fmol/mg protein/nM, respectively. The data suggest that α₂-adrenoceptors exist in rat brain cortex as homodimers.     

表皮穿透和GABAA受体不敏感性在小菜蛾对阿维菌素抗性中的作用

用氚标记阿维菌素点滴处理阿维菌素敏感（ABM-S）和抗性（ABM-R）种群小菜蛾幼虫,结果显示,在5～360 min内的7个不同处理时间,ABM-R种群的平均表皮穿透量比ABM-S种群少1.5倍,处理24 h后,ABM-R种群仍有45.9%的3H-阿维菌素滞留于体表,而ABM-S种群却有98.4%的药剂穿透表皮。放射配体结合分析表明,GABAA受体结合性质的改变是小菜蛾对阿维菌素的另一抗性机制,ABM-S种群（K_d=10.9368±0.4374 nmol/L）和ABM-R种群（K_d=9.8328±0.3933　nmol/L）的受体亲和力无显著差异,但抗性种群的最大结合量（B_max=71.2842±4.9910　fmol/mg 蛋白）比敏感种群（B_max=112.0255±7.8418　fmol/mg 蛋白）降低63.6%,即抗性是受体数目的减少而非结构上的改变。     

Characterization of the Binding of the GABA Agonist [3H]Piperidine-4-Sulphonic Acid to Bovine Brain Synaptic Membranes

Abstract: The binding of radioactive piperidine-4-sulphonic acid ([³H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0°C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: K_D= 17 ± 7 nM (B_max= 0.15 ± 0.07 pmol/mg protein) and K_D= 237 ± 100 nM (B_max= 0.80 ± 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [³H]P4S. The slow binding component (k_on= 5.6 × 10⁷ or 8.8 × 10⁷ M^-1 min^-1, k_Off= 0.83 min^-1, and K_D= 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing potassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (K_D= 11 nM, B_max= 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [³H]P4S appears to involve the same two populations of sites with B_max values similar to those for [³H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [³H]P4S and [³H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.     

CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

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.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Differential distribution of endothelin peptides and receptors in human adrenal gland

Summary Sub-type selective ligands revealed a differential distribution of endothelin (ET) receptors within human adrenal glands. High densities of ET_A receptors were localized, using [¹²⁵I]-PD151242, to the smooth muscle layer of the arteries, smaller vessels within the capsular plexus and to the secretory cells of zona glomerulosa (K _D=139.8±39.7,B _max=69.7±9.1 fmol mg⁻¹ protein, mean of 3 individuals±sem). ET_B receptors were present in the medulla (K _D=145.2±16.4,B _max=75.5±12.3), zona glomerulosa (K_D=100.6±35.1,B _max=63.1±10.0), fasiculata (K _D 145.1±162.,B _max=67.9±6.9) and reticularis (K_D=118.2±18.6,B _max=71.9±6.5). ET_B receptors were not detected within the smooth muscle of the vasculature. Messenger RNA encoding both sub-types was present in adrenals. ET-like immunoreactivity was localized to the cytoplasm of the endothelial cells from arteries supplying the gland and resistance vessels within the capsular plexus. Staining was also detected in these cells using anti-big ET-1 and less intensely with anti-big ET-2 antisera but not within cells within the cortex or medulla. Big ET-3-like immunoreactivity was localized to secretory cells of the medulla. Staining was not found using antiserum that could detect ET-3, suggesting further processing of big ET-3 may occur within the plasma, and that the cdrenals could be a source of ET-3. The presence of ET-1 was confirmed by high performance liquid chromatography and radioimmunoassay although ET-3 was not detected. The results suggest that ET-1 is the predominant mature isoform, which is localized mainly to adrenal vasculature, particularly the capsular plexus, and may contribute to blood flow regulation in the gland.     

d-Aspartate binding sites in rat Harderian gland

Radioligand binding of d-[³H]aspartic and l-[³H]glutamic acids to plasma membranes from rat Harderian gland was evaluated. Binding was optimal under physiological conditions of pH and temperature, and equilibrium was reached within 50 min. Specific binding for d-Asp and l-Glu was saturable, and Eadie–Hofstee analysis revealed interaction with a single population of binding sites (for d-Asp K _d = 860 ± 28 nM, B _max = 27.2 ± 0.5 pmol/mg protein; for l-Glu, K _d = 580 ± 15 nM and B _max = 51.3 ± 0.8 pmol/mg protein). l-[³H]glutamate had higher affinity and a greater percentage of specific binding than did d-[³H]aspartate. The pharmacological binding specificity of l-[³H]glutamate indicated an interaction with NMDA-type receptors. Specifically, the order of potency of the displacing compound tested was l-Glu > d-Asp > NMDA > MK801 > d-AP5 > glycine. For d-[³H]aspartate, the data revealed an interaction of d-Asp with either NMDA-type receptors or putative specific binding sites.     

[125I]RTI-55 Binding to Cocaine-Sensitive Dopaminergic and Serotonergic Uptake Sites in the Human Brain

[¹²⁵I]RTI-55 is a newly synthesized cocaine congener that may offer advantages over other ligands previously used to examine cocaine binding sites. However, the in vitro pharmacological and anatomical characterization of [¹²⁵I]RTI-55 binding sites has not been previously performed in human brain. To determine the specificity, stability, and feasibility of [¹²⁵I]RTI-55 for use in radioligand binding assays in postmortem human tissue, a series of experiments were performed characterizing [¹²⁵I]RTI-55 binding sites in human brain using homogenized membrane preparations and quantitative autoradtography. Analysis of the association, dissociation, and saturation data favored two-phase processes. A curve-fitting analysis of the data derived in saturation experiments found a high-affinity site with K_D= 66 ± 35 pM and S_max= 13.2 ± 10.1 pmol/g of tissue and a low-affinity site with K_D= 1.52 ± 0.55 nM and B_max of 47.5 ± 11-2 pmol/g of tissue. Competition by ligands known to bind to the dopamine transporter showed a rank order of RTI-55 > GBR-12909 > mazindol > WIN 35428 > = methylphenidate > (?)-cocaine > buproprion > (±)-amphetamine. Binding to serotonergic sites was evaluated in the midbrain. Results of the saturation experiment performed autoradiographically in the midbrain showed a single site with K_D= 370 ± 84 pM. It appears that [¹²⁵I]RTI-55 should be useful in further studies of the regulation of cocaine binding sites using postmortem human specimens.     

Binding of β1-adrenoreceptor antagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial nodeantagonist [3H]CGP 26505 in rat cerebral cortex and sinus atrial node

Specific β₁-adrenoreceptors antagonist [³H]CGP 26505 binding was characterized in rat cerebral cortex and heart sinus atrial node. In both tissues [³H]CGP 26505 binding was maximal at 25°C, it was specific, saturable and protein concentration dependent. Scatchard analysis of saturation isotherms of specific [³H]CGP 26505 binding in cerebral cortex showed that [³H]CGP 26505 binds a single class of high affinity sites with a dissociation constant (K_D) of 1±0.3 nM and a maximal number of binding sites (B_max) of 40±2 fmol/mg of protein. In sinus atrial node, [³H]-CGP 26505 binds a single class of high affinity sites (K_D=1.9±0.4 nM, B_max=28±2 fmol/mg of protein).     

BINDING OF [99mTc]CHONDROITIN SULFATE TO SCAVENGER RECEPTORS ON HUMAN CHONDROCYTES AS COMPARED TO BINDING OF OXIDIZED [125I]LDL ON HUMAN MACROPHAGES

ABSTRACT

Chondroitin sulfate (CS) used for treatment of osteoarthritis exerts distinct effects on human articular chondrocytes in vitro. We performed a binding analysis with ^99mTc-labeled CS (Condrosulf, a commercial CS preparation containing calcium stearate) and cultured human chondrocytes in order to evaluate the presence of specific receptors. Saturation binding at 37°C for 2?h revealed the presence of high-affinity binding sites for CS with a K_d of 2.3 × 10^?9?mol/L and a B_max of 5.0 × 10⁸. Extensive dialysis of Chondrosulf led to a decrease of the binding affinity by 52.5 ± 19.5% and of the number of CS binding sites/cell by 62.0 ± 14.0%, demonstrating that the additive present in the Condrosulf preparation enhances CS binding. The nature of the binding site is not yet known but evidence exists in the literature that the scavenger receptor CD36, thoroughly investigated on macrophages, is also found on chondrocytes and might be involved in CS binding. Therefore, we undertook a comparative binding study with human monocytes and labelled LDL and oxidized LDL, the latter being a postulated atherogenic agent in atherosclerosis. For [¹²⁵I]-LDL binding we found a K_d of 0.45 × 10^?8?mol/L and a B_max of 0.14 × 10⁶ on quiescent monocytes and for [¹²⁵I]-(ox)LDL binding a K_d of 1.8 × 10^?8?mol/L and a B_max of 1.3 × 10⁶ using LPS-activated monocytes. These data are comparable to the binding affinity found for lipoprotein–proteoglycan-complexes and hence are an indication but not a proof that CD36 is involved in CS binding to human chondrocytes.     

Day–night specific binding of 2-[125I]Iodomelatonin and melatonin content in gill, small intestine and kidney of three fish species

Some of melatonin’s (Mel) well-established physiological effects are mediated via high-affinity cell-membrane receptors belonging to the superfamily of G-protein-coupled receptors. Specific binding of ligand 2-[¹²⁵I]iodomelatonin, using membrane preparations from osmoregulatory tissues of flounder, rainbow trout and sea bream, together with Mel concentrations in the tissues and plasma were studied. The kidney, gill and small intestine samples were collected during the day and at night. The dissociation constants (K _d) and maximal binding densities (B _max) were calculated for each tissue at 11:00 and 23:00 h. The binding sites with K _d values in the tissues in the picomolar range indicated the high affinity. K _d and B _max values were tissue- and species-dependent. The GTP analogue [Guanosine 5′-O-(3-thiotriphosphate)] treatment significantly reduced the B _max value, indicating that the 2-[¹²⁵I]iodomelatonin-binding sites are probably coupled to a G-protein. No daily variations in K _d and B _max values were observed. These are the first studies of the presence of 2-[¹²⁵I]iodomelatonin-binding sites in the small intestine, kidney tubule and gill of fish. The data strongly suggest new potential targets for Mel action and the influence of Mel on water/ion balance in fish. The intestine seems to be a site of Mel synthesis and/or an active accumulation of the hormone.     

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.     

A COMPARATIVE STUDY ON THE SYNTHESIS OF THE NATRIURETIC HORMONE DOPAMINE IN OK AND LLC-PK1CELLS

TheV_maxvalues (in nmol/mg protein/15 min) for AAAD in OK cells (0.94±0.08) were found to be significantly (P<0.01) lower than those observed in LLC-PK₁cells (4.37±0.08). However, in both cell lines decarboxylation reaction was a saturable process with similarK_mvalues (OK cells=1.1 mm (0.3, 1.8); LLC-PK₁cells=1.8 mm (1.6, 2.1)). Contrariwise to OK cells, decarboxylation ofl -DOPA to dopamine in LLC-PK₁cells followed a linear (7.6±0.1 pmol/mg protein/min) non-saturable kinetics till 120 min of incubation. The formation of dopamine from increasing concentrations ofl -DOPA (10 to 500 μm ) followed a non-linear kinetics in both cell lines; the process ofl -DOPA decarboxylation was saturated at low concentrations ofl -DOPA with an apparentK_mvalue of 11 μm (0.2, 22.6) in OK cells and 27.4 μm (11.1, 43.7) in LLC-PK₁cells. The formation of dopamine in LLC-PK₁cells (V_max=2097±113 pmol/mg protein/6 min) was 13.7-fold that occurred in OK cells (V_max=153±10 pmol/mg protein/6 min). In conclusion, LLC-PK₁cells appear to be endowed with a greater ability to form dopamine from exogenousl -DOPA when compared to OK cells.     

兔肝细胞膜高密度脂蛋白受体酶联免疫检测法的研究

用辣根过氧化物酶标记羊抗人apoAI-IgG,建立了检测兔肝细胞膜HDL受体的酶联免疫吸附检测法.测定时, HDL结合量按抗体-配体-抗配体抗体酶交联物反应制作的标准曲线确定;膜蛋白非特异吸附则用与酶交联的抗体来源相同的同种动物血浆HDL平行抑制试验消除.实验测得正常家兔肝细胞膜HDL受体K_d值为7.17±1.18 mg/L,B_max值为(622.5±146.1)mg/g(n=7).     

β-Adrenoreceptor agonists and antagonists modulate the activity of α<Subscript>2</Subscript>-adrenoreceptors in rat cortex cerebral membranes

The influence of isoprenaline- and propranolole-induced activation and inhibition of β-adrenoreceptors on the specific nonselective α₂-antagonist [³H]RX821002 binding was studied on rat cerebral cortex subcellular membrane fractions. It was shown that the ligand-receptor interaction for α₂-adrenoreceptors corresponded to the model that assumed the presence of one receptor pool and binding of two ligand molecules to a receptor dimer. The following parameters were determined for [³H]RX821002 binding to α₂-adrenoreceptors: K _d1 = 1.57 ± 0.27 nM, B _max = 7.24 ± 1.63 fmol/mg of protein, n = 2. In the case of isoprenaline-induced activation of β-adrenoreceptors the binding of radiolabeled ligand to α₂-adrenoreceptors was described by the same model. The affinity of α₂-adrenoreceptors for [³H]RX821002 decreased more than twofold (K _d = 3.55 ± 0.02 nM) and the quantity of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg of protein). Propranolole changed the model of ligand binding, and two pools of receptors were detected with the following parameters: K _d1 = 0.61 ± 0.02 nM, K _d2 = 3.41 ± 0.13 nM, B _ml = 1.88 ± 0.028 fmol/mg of protein, B _m2 = 9.27 ± 0.08 fmol/mg of protein, n = 2. The data suggest that α₂-adrenoreceptors in subcellular membrane fractions from rat cerebral cortex exist in dimeric form. Isoprenaline and propranolole exhibit modulating effect on the specific antagonist binding to α₂-adrenoreceptors, which results in the inhibition and alteration of [³H]RX821002 binding parameters.     

d-glucose uptake in human liver cell cultures

Summary The kinetic parameters ford-glucose uptake were studied in human liver cell cultures under strictly defined experimental conditions. Using a wide concentration range (0.005 to 30 mmol/l), the kinetic data obtained suggested strongly thatd-glucose in human liver cell cultures can be transported by two separate systems. For the high-affinity system, the apparentK _m was 0.645±0.21 mmol/l and the V_max, 12.49±3.74 nmol/mg protein per min. For the low-affinity system, the apparentK _m was 6.91±0.58 mmol/l and the V_max, 79.90±5.27 nmol/mg protein per min. At a concentration of 2.1×10⁻⁷ mol/l, cytochalasin B preferentially inhibited the high-affinityd-glucose site or transport system. The time course ofd-glucose uptake, studied in two cell lines from patients with hereditary fructose intolerance, was significantly higher than for the control lines. This work was supported by Grant I.N.S.E.R.M. CRL 77-5-210-4.     

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.     

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.     

Endothelin-1 Binding to Endothelin Receptors in the Rat Anterior Pituitary Gland: Interaction in the Recognition of Endothelin-1 Between ETA and ETB Receptors

1. ¹²⁵I-Endothelin (ET)-1 binding to the rat anterior pituitary gland was saturable and single, with a K _d of 71 pM and a B _max of 120 fmol/mg.2. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, the binding parameters were 8.3 pM and 8.0 fmol/mg, whereas 10 nM sarafotoxin S6c (ET_Bagonist) exerted little change in these binding parameters (K _d,72pM;B _max, 110 fmol/mg).3. ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620, and BQ-788 competitively inhibited ¹²⁵I-ET-1 binding, only when 1.0 M BQ-123 was present in the incubation buffer.4. Thus, the ET_B receptor is capable of binding ET-1 when the ET_A receptor is being occupied by BQ-123. A collaboration mechanism between the ET_A and the ET_B receptor may function in the recognition of ET-1, a typical bivalent ligand.