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 [3H] γ-Aminobutyric Acid and [3H]Muscimol in Purified Rat Brain Synaptic Plasma Membranes and the Effects of Bicuculline

Abstract: The binding of [³H] γ-aminobutyric acid ([³H]GABA) and [³H]muscimol has been studied in purified synaptic plasma membrane (SPM) preparations from rat brain. Scatchard analysis of specific binding (defined as that displaced by 100 μMγ-aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent K_D and B_max values for [³H]GABA at 4°C were K_D1, 20 nM; K_D2,165 nM; B_max1, 0.48 pmol;B_max2, 6.0 pmol. mg^?1; for [³H]muscimol at 4°C they were: K_D1, 1.75 nM; K_D2, 17.5 nM; B_maxl, 0.84 pmol. mg^?1; B_max2, 4.8 pmol.mg^?1; and for [³H]muscimol at 37°C they were: K_D1, 7.0 nM; K_m, 60 nM; B_max], 0.5 pmol-mg^?1; B_max2, 7.2 pmol-mg¹. Under the experimental conditions used, the similar B_milx values for [³H]GABA and [³H]muscimol binding to the SPM preparations suggests that the high- and low-affinity components for the two radiolabeled ligands are identical. The effects of the GAB A antagonist bicuculline on the binding of [³H]muscimol at 4^CC and 37°C were studied. At 4°C, antagonism of muscimol binding appeared to be competitive at the high-affinity site but noncompetitive at the low-affinity site. At 37°C, antagonism was again competitive at the high-affinity site but was of a mixed competitive/noncompetitive nature at the low-affinity site. Assuming that binding to the high-affinity site is associated with the pharmacological actions of bicuculline, the apparent K_D values obtained suggest a pA₂ value of 5.3 against [³H]muscimol at 4°C and 37°C. This figure is in good agreement with several estimates of the potency of bicuculline based on pharmacological measurements. Results from displacement studies using [³H]GABA and [³H]muscimol suggest that [³H]GABA might be a more satisfactory ligand than [³H]muscimol in GABA radioreceptor assays.     

[3H] verapamil binding sites in skeletal muscle tranverse tubule membranes

[³H]verapamil binding to muscle tubule membrane has the following properties. K_D = 27 ± 5 nM and maximum binding capacity B_max = 50 ± 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [³H]verapamil versus [³H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k₁) and dissociation (k_?1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [³H]verapamil binding with K_D values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [³H]verapamil binding with half-maximum inhibition values (K_0.5) between 1 and 5 nM.     

[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.     

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.     

Leukotriene C4 ([3H] - LTC4) binding to membranes isolated from a hamster smooth muscle cell line (DDTIMF2)

Leukotriene C₄ (LTC₄) has been demonstrated to induce contraction of the smooth muscle cell line DDTIMF2. A partially purified membrane fraction obtained from these cells exhibited a high affinity binding site for LTC₄. Binding of [³H]-LTC₄ was saturable, specific and reversible with a dissociation constant (K_d) of 21 ± 4 nM. The maximum number of binding sites (B_max) was 55 ± 5 pmol/mg of protein. Specificity was demonstrated in competition studies in which the Ki of LTC₄ against specifically bound [³H] - LTC₄ was 12 nM whereas Leukotriene D₄ (LTD₄) and Leukotriene E₄ (LTE₄) had a Ki of 38 ± 4 and 4.7 ± 0.5 nM respectively. A previously described antagonist of leukotriene-induced smooth muscle contraction PFL 55712 had a K_i of 23 ± 2 nM as determined by competition binding experiments.     

The Binding of the GAB A Agonist [3H]THIP to Rat Brain Synaptic Membranes

Abstract: THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a specific GABA agonist with potent analgesic properties. The binding of radioactive THIP to thoroughly washed, frozen, and thawed membranes isolated from rat brains has been studied at 2°C under sodium ion-free conditions and compared with the binding of [³H]GABA and [³H]piperidine-4-sulphonic acid ([³H]P4S). The best computer fits to the experimental data were in all cases attained with a receptor model based on three independent binding sites, of which only the high- and medium-affinity sites could be characterised satisfactorily. While the K_D values were found to be comparable for all three ligands employed, the density of the high-affinity binding site (B_M1) was, with the exception of the membranes from the cerebellum, considerably lower for [³H]THIP than for [³H]GABA and [³ H]P4S. The regional distribution of the GABA receptors, which bind [³H]THIP, was different from those recognizing [³H]GABA and [³H]P4S. A number of analogues, including asymmetric compounds with known configuration, were tested as inhibitors of the binding of [³H]GABA, [³H]muscimol, [³H]THIP, [³H]isoguvacine, and [³H]P4S. The concentrations of the asymmetric compounds required for the inhibition of [³H]P4S binding were much higher than those required for the displacement of [³H]GABA, [³H]muscimol, [³H]THIP, and [³H]isoguvacine. The comparable relative potencies of inhibitors do, however, indicate that all of the ligands bind to the GABA receptors.     

Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [³H]glycine and [³H]strychine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na⁺-independent. [³H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with K_D=12 and 82 nM and B_Max=3.1 and 3.06 pmol/mg protein respectively. [³H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 K_D values were 3.9 and 18.7 nM, and B_Max values were 1.1 and 7.1 pmol/mg protein, respecitively. Membranes from the P2 synaptosomal fraction showed K_D's of 0.6 and 48 nM and B_Max's of 0.4 and 4.5 pmol/mg. Specific [³H]glycine binding was displaced by -alanine, l-serine, d-serine and HA966, but not by strychnine 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [³H]strychnine, binding was partially displaced by glycine and related aminoacids and totally displaced only by 2-NH₂-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.     

Solubilization of High-Affinity, Guanine Nucjeotide-Sensitive μ-Opioid Receptors from 7315c Cell Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [³H]-etorphine with high affinity (K_D= 0.304 ± 0.06 nM; B_max= 154 ± 33 fmol/mg of protein). Of the membrane-associated [³H]etorphine binding sites, 40 ± 5% were recovered in the solubilized fraction. Both μ-selective and non-selective enkephalins competed with [³H]etorphine for the solubilized binding sites; in contrast, 5- and K-opioid enkephalins failed to compete with [³H]etorphine for the solubilized binding sites at concentrations of <1 μM.The μ-selective ligand [³H][D-Ala²,A/-Me-Phe⁴,Gly⁵-ol]enkephalin also bound with high affinity (K_D= 0.79 rM; B_max= 108±17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin binding sites, 43 ± 3% were recovered in the solubilized material. Guanosine 5′-O-(3-thiotriphosphate), GTP, and guanosine 5′-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin binding in a concentration-dependent manner. Finally, μ-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate.     

The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [³H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [³H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a K_d of 113 nM and B_max of 0.65 pmol/10⁶ cells in freshly isolated cell suspension and K_d of 1.65 μM and B_max of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [³H]PAF itself was found in the same cell preparations. The binding of [³H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K_i of 3.8 nM and B_max of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam also competed for the [³H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [³H]WEB 2086 with a rank order BN 50739⪢Ro 5-4864≥clonazepam. Interestingly, bicuculline, a specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [³H]WEB 2086. The binding of [³H]flunitrazepam was inhibited with a rank potency BN 50739⪢WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

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.     

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.     

Specific binding of [3H]nitrendipine to membranes from coronary arteries and heart in relation to pharmacological effects. Paradoxical stimulation by diltiazem

High affinity binding sites for the calcium channel inhibitor [³H]nitrendipine have been identified in microsomes from pig coronary arteries (K_D=1.6 nM; B_max=35 fmol/mg) and in purified sarcolemma from dog heart (K_D=0.11 nM; B_max=230 fmol/mg). [³H]nitrendipine binding to coronary artery microsomes was completely inhibited by nifedipine, partially by verapamil and D600 and, surprisingly, was stimulated by d-cis-diltiazem but not by 1-cis-diltiazem, a less active isomer. Half-maximal relaxation of KCl-depolarized coronary rings occurred in a slow process at 1 nM nitrendipine or 100 nM d-cis-diltiazem. In dog trabecular strips, nitrendipine caused a negative inotropic response (ED₅₀=1μM). These results suggest that there may be multiple binding sites for different “subclasses” of calcium channel inhibitors, and that drug binding sites may be different molecular entities from the putative calcium channels.     

Solubilization of High-Affinity,Guanine Nucleotide-Sensitive μ-Opioid Receptors from Rat Brain Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate appeared to stabilize the μ-opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [³H][d -Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin ([³H]DAMGO), a μ-selective opioid agonist, with high affinity (K_D = 1.90 ± 0.93 nM; B_max = 629 ± 162 fmol/mg of protein). Of the membrane-associated [³H]-DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [³H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate). The solubilized receptor also bound [³H]diprenorphine, a nonselective opioid antagonist, with high affinity (K_D = 1.4 ± 0.39 nM, B_max = 920 ± 154 fmol/mg of protein). Guanosine 5′-O-(3-thiotriphosphate) did not diminish [³H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [³H]diprenorphine for the solubilized binding sites; in contrast, [d -Pen²,d -Pen⁵]-enkephalin, a δ-selective opioid agonist, and U50488H, a κ-selective opioid agonist, failed to compete with [³H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [³H]diprenorphine binding sites better fit a two-site than a one-site model with K_Dhigh = 2.17 ± 1.5 nM, B_max = 648 ± 110 fmol/mg of protein and K_Dlow = 468 ± 63 nM, B_max = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with K_D = 815 ± 33 nM, B_max = 965 ± 124 fmol/mg of protein.     

An [3H]oxotremorine binding method reveals regulatory changes by guanine nucleotides in cholinergic muscarinic receptors of cerebral cortex

A rapid, reliable filtration method for [³H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [³H]Oxotremorine binds to cerebral cortex membranes with high affinity (K _D, 1.9 nM) and low capacity (B _max, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [³H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value ofK _D (2.3 nM). Displacement studies with 1–4000 nM oxotremorine showed the existence of a second binding site of low affinity (K _D, 1.2 M) and large capacity (B _max, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [³H]oxotremorine binding with an IC 50 of 0.3 M. Saturation assays, in the presence of 0.5 M Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.     

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.     

[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

Decreased GABA<Subscript>A</Subscript> Receptor Function in the Brain Stem during Pancreatic Regeneration in Rats

Gamma amino butyric acid is a major inhibitory neurotransmitter in the central nervous system. In the present study we have investigated the alteration of GABA receptors in the brain stem of rats during pancreatic regeneration. Three groups of rats were used for the study: sham operated, 72 h and 7 days partially pancreatectomised. GABA was quantified by [³H]GABA receptor displacement method. GABA receptor kinetic parameters were studied by using the binding of [³H]GABA as ligand to the Triton X-100 treated membranes and displacement with unlabelled GABA. GABA_A receptor activity was studied by using the [³H]bicuculline and displacement with unlabelled bicuculline. GABA content significantly decreased (P < 0.001) in the brain stem during the regeneration of pancreas. The high affinity GABA receptor binding showed a significant decrease in B _max (P < 0.01) and K _d (P < 0.05) in 72 h and 7 days after partial pancreatectomy. [³H]bicuculline binding showed a significant decrease in B _max and K _d (P < 0.001) in 72 h pancreatectomised rats when compared with sham where as B _max and K _d reversed to near sham after 7 days of pancreatectomy. The results suggest that GABA through GABA receptors in brain stem has a regulatory role during active regeneration of pancreas which will have immense clinical significance in the treatment of diabetes.     

Labelling of 5-Hydroxytryptamine3 Receptors with a Novel 5-HT3 Receptor Ligand, [3H]RS-42358–197

Abstract: RS-42358–197{(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1H-benzo[de]isoquinolin-1-one hydrochloride} displaced the prototypic 5-hydroxytryptamine₃ (5-HT₃) receptor ligand [³H]quipazine in rat cerebral cortical membranes with an affinity (pK_i) of 9.8 ± 0.1, while having weak affinity (pK_i < 6.0) in 23 other receptor binding assays. [³H]RS-42358–197 was then utilized to label 5-HT₃ receptors in a variety of tissues. [³H]RS-42358–197 labelled high-affinity and saturable binding sites in membranes from rat cortex, NG108–15 cells, and rabbit ileal myenteric plexus with affinities (K_D) of 0.12 ± 0.01, 0.20 ± 0.01, and 0.10 ± 0.01 nM and densities (B_max) of 16.0 ± 2.0, 660 ± 74, and 88 ± 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [³H]RS-42358–197 was similar to that labelled with [³H]GR 65630, but was significantly less than that found with [³H]-quipazine. The binding of [³H]RS-42358–197 had a pharmacological profile similar to that of [³H]quipazine, as indicated by the rank order of displacement potencies: RS-42358–197 > (S)-zacopride > tropisetron > (R)-zacopride > ondansetron > MDL72222 > 5-HT. However, differences in 5-HT₃ receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1-(m-chlorophenyl)biguanide when displacing [³H]RS-42358-197 binding. [³H]RS-42358–197 also labelled a population (B_max= 91 ± 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (K_D= 1.6 ± 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5-HT₃ receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [³H]RS-42358–197 have detected differences in 5-HT₃ receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5-HT₃ receptor.     

Specific binding sites for corticosterone in isolated cells and plasma membrane from rat liver

Summary The specific binding of [³H]corticosterone to hepatocytes is a nonsaturable, reversible and temperature-dependent process. The binding to liver purified plasma membrane fraction is also specific, reversible and temperature dependent but it is saturable. Two types of independent and equivalent binding sites have been determined from hepatocytes. One of them has high affinity and low binding capacity (K _D=8.8nm andB _max=1477 fmol/mg protein) and the other one has low affinity and high binding capacity (K _D=91nm andB _max=9015 fmol/mg). In plasma membrane only one type of binding site has been characterized (K _D=11.2nm andB _max=1982 fmol/mg). As it can be deduced from displacement data obtained in hepatocytes and plasma membrane the high affinity binding sites are different from the glucocorticoid, progesterone nuclear receptors and the Na⁺,K⁺-ATPase digitalis receptor. Probably it is of the same nature that the one determinate for [³H]cortisol and [³H]corticosterone in mouse liver plasma membrane. Beta-and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [³H]corticosterone binding to hepatocytes and plasma membranes; therefore, these binding sites are independent of adrenergic receptors. The binding sites in hepatocytes and plasma membranes are not exclusive for corticosterone but other steroids are also bound with very different affinities.