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.     

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.     

Ecdysteroid receptors in a tumorous blood cell line of Drosophila melanogaster

The tumorous Drosophila melanogaster blood cell line BII has been studied for evidence for the presence of ecdysteroid receptors. The [³H]ponasterone A (pon A)* used in this study has been extensively purified, and the location of the tritium in the molecule has been partially determined. BII cells do not metabolise ecdysteroids. Intact cells demonstrate a considerable specific uptake of [³H]pon A which is saturable, apparently showing two specific components: a very high affinity component (K_D = 0.3 nM) and a high affinity component (K_D = 2 nM). The specific binding of [³H]pon A to whole cells is compatible with unlabelled ecdysteroids, but not with mammalian steroid hormones. The association rate constant (k_a) for [³H]pon A was determined to be 3 × 10⁷M^?1min^?1 at 21 °C, while the dissociation rate constant (k_d) for the specifically bound [³H]pon A was found to be 4.4 × 10^?3/min. Together, the kinetic rate constants yield a value of 0.15 nM for the K_D. The receptors have been partially characterised in a cell-free extract prepared by sonification of the cells. The optimum pH for extraction and hormone binding is 8.2. Scatchard plots of binding data indicate that the cell-free extract also contains two high affinity specific binding components (k_D = 0.1 nM and K_D = 1 nM). The hgih affinity binders are macromolecular, as shown by chromatography on Sephadex G-25, and are susceptible to protease digestion, heat, and treatment with N-ethylmaleimide. Sucrose density centrifugation of the labelled receptor shows one peak at approximately 6S. The stability of the receptor preparation has been studied and conditions have been empirically determined (10% w/v sucrose, 25 mM dithioerthreitol, and 10 mM citrate), whereby the binding capacity of the unlabelled receptor is stable for at least 8 weeks if frozen at ?20°C.     

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).     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.     

Identification of stereospecific [3H]spiroperidol binding sites in mammalian lymphocytes

Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [³H]spiroperidol. The specific binding is saturable with two components (K_D1 = 1.9 nM, K_D2 = 36.2 nM). Determination of the K_D by kinetic studies measuring rate constants for association and dissociation provided K_D values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [³H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (?)butaclamol. The relative potencies of different antidopaminergic agents in competing for [³H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response.     

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.     

Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

Characterization of binding of [3H]PD 128907, A selective Dopamine D3 receptor agonist ligand,to CHO-K1 cells

PD 128907 [4a R, 10 b R-(+)-trans- 3, 4, 4a, 10 b - tetrahydro - 4- n-propy12 H,5H-[1] benzopyrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (K_i, 1 nM) versus human D₂ receptors (K_i, 1183 nM) and a 10000-fold selectivity versus human D₄ receptors (K_i, 7000 nM) using [³H]spiperone as the radioligand in CHO-K1-cells. Studies with [³H]PD 128907, showed saturable, high affinity binding to human D₃ receptors expressed in CHO-K1 cells (CHO-K1-D₃) with an equilibrium dissociation constant (K_d) of 0.99 nM and a binding density (B_max) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D₂ receptors (CHO-K1-D₂). The rank order of potency for inhibition of [³H]PD 128907 binding with reference DA agents was consistent with reported values for D₃ receptors. These results indicate that [³H]PD 128907 is a new, highly selective D₃ receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D₃ receptors.     

Specific calcium antagonist binding sites in brain

The binding of the calcium antagonist [³H] nitrendipine ([³H] NDP) to brain and heart is described and the brain site is characterized. The binding is saturable, specific and of very high affinity with K_D values of 0.16 nM in brain and 0.21 nM in heart. Our kinetic results are similar to those recently reported by two other groups (1,2), indicating a saturable, high affinity binding site in brain. In brain the binding sites are enriched in crude nuclear and synaptosomal fractions. The highest levels of binding are seen in the hippocampus, caudate and cerebral cortex with much lower levels in the cerebellum and pons. Calcium has a marked stimulatory effect on [³H] NDP binding at 10^?4 M. Addition of 0.5 mM CaCl₂ to EDTA treated membranes nearly doubles the number of binding sites. Of the many drugs and neurotransmitters tested only other calcium antagonists, i.e., verapamil, inhibit binding (IC₅₀ = 250 nM). The inhibition of [³H] NDP binding by verapamil is apparently non-competitive and not complete, suggesting that [³H] NDP binds to several sites, only some of which are inhibited by verapamil. The [³H] NDP binding site is probably a protein since it is very sensitive to trypsin, heat and sulfhydryl reagents.     

[3H]muscimol binding in the rat retina

Crude membrane fractions were prepared from rat retinae and used to study the specific binding of [³H]muscimol, a potent GABA agonist. Specific [³H]muscimol binding was enhanced 2–3 fold by pretreatment of the membranes with 0.025% Triton X-100. Two muscimol binding sites were demonstrated with K_D values of 4.4 and 12.3 nM. GABA, muscimol, and 3-aminopropanesulfonic acid were the most potent inhibitors of specific [³H]muscimol binding with K_I values of 15, 10, and 50 nM, respectively. These data are consistent with binding to the synaptic GABA receptor.     

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 oligopeptide chemotactic factor receptor on human polymorphonuclear leukocyte membranes exists in two affinity states

The binding of the chemoattractant N-formyl-methionylleucyl-[³H]phenylalanine to intact polymorphonuclear leukocytes and membrane preparations was analyzed by computer methods. Whole viable cells bind the chemoattractant with a single dissociation constant (K_D) of 22.3 ± 2.4 nM and contain an average of 55,000 receptors percell. In contrast, the binding data using membrane preparations were consistent with the presence of two classes of binding sites with average K_Ds of 0.53 ± 0.01 nM and 24.4 ± 1.2 nM. The high affinity receptors accounted for ca. 25% of the binding sites. Increasing the receptor occupancy did not affect the rate of dissociation of the ligand-receptor complex thus negative cooperativity is not a likely explanation for the complex binding isotherms. On the other hand, the dissociation kinetics did agree with the two affinity receptor model.     

A calcium antagonist drug binding site in skeletal muscle sarcoplasmic reticulum: Evidence for a calcium channel

The sarcoplasmic reticulum (S.R.) of rabbit skeletal muscle has been found to contain a single, high affinity binding site for the Ca antagonist drug [³H] -nitrendipine. Two subfractions of the reticulum were studied, the heavy (HSR) and light (LSR) preparations, which exhibited similar nitrendipine equilibrium dissociation constants (K_D) of 1nM. Crude cardiac and brain membranes assayed under the same conditions exhibited K_D values of 0.2–0.3nM. The concentration of binding sites per mg. protein (B_max) in HSR was found to be very high, namely 6.7 picomoles/mg, some four times greater than that of LSR. [³H] -nitrendipine binding to HSR was reversible and inhibited by the Ca antagonists flunarizine and verapamil, and by the intracellular Ca release antagonist TMB-8 (8-diethylamino-octyl 3,4,5- trimethylbenzoate hydrochloride). However, unlabelled nitrendipine at 2 × 10^?5M had no effect on contraction of isolated electrically stimulated rabbit lumbrical or rat diaphragm muscles, nor did it affect the neuromuscular junction as studied in rat phrenic nerve-diaphragm preparations. Also, little effect of 2 × 10^?5M nitrendipine was seen on net ⁴⁵Ca uptake by HSR. These results suggest that [³H] -nitrendipine binding to skeletal muscle S.R. resembles that of brain membranes, which also contain a high affinity binding site for [³H] -nitrendipine and which similarly are pharmacologically insensitive to this dihydropyridine type of Ca channel blocking agent. Since HSR is also enriched in calsequestrin and terminal cysternae from which Ca is released in vivo, it seems likely that the [³H]- nitrendipine binding sites in S.R. are associated with Ca channels in the S.R.     

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.     

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.     

Characteristics of Binding of [3H]WAY100635 to Rat Hippocampal Membranes

Kinetic analysis of binding of [³H][N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide ([³H]WAY100635) to 5-HT_1A receptors in rat hippocampal membranes has revealed complex regulation mechanism for this radioligand. Saturation binding experiments revealed that [³H]WAY100635 binds to a single class of receptors with very high apparent affinity (K _D = 87 ± 4 pM, B _max = 15.1 ± 0.2 fmol/mg protein). The binding was almost irreversible, as the dissociation rate constant obtained k _off = (7.8 ± 1.1) × 10⁻³ min⁻¹, means that equilibrium with this radioligand cannot be achieved before 7.5 h incubation at 25°C. Systematic association kinetic studies of [³H]WAY100635 binding revealed sharp reaction acceleration at higher radioligand concentration, proposing mechanism of positive cooperativity. The affinities of antagonists determined from competition with [³H]WAY100635 did not coincide with their abilities to inhibit 5-HT-dependent activation of [³⁵S]GTPγS binding probably due to the ligand’s kinetic peculiarities. Thus, [³H]WAY100635 appears to be an excellent tool for determining receptor binding sites, but its applicability in equilibrium studies is strongly limited.     

Involvement of carboxyl groups in chloride transport and reversible DIDS binding to band 3 protein in human erythrocytes

Noncovalent DIDS binding to Band 3 (AE1) protein in human erythrocyte membranes, modified by non-penetrating, water soluble 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (EAC), was studied at 0°C in the presence of 165 mM KCl. Under experimental conditions applied up to (48 ± 5) % of irreversible chloride self-exchange inhibition was observed. The apparent dissociation constant, KD, for “DIDS-Band 3” complex, determined from the chloride transport experiments, was (34 ± 3) nM and (80 ± 12) nM for control and EAC-treated resealed ghosts, respectively. The inhibition constant, Ki, for DIDS was (35 ± 6) nM and (60 ± 8) nM in control and EAC-treated ghosts, respectively. The reduced affinity for DIDS reversible binding was not a result of negative cooperativity of DIDS binding sites of Band 3 oligomer since Hill’s coefficients were indistinguishable from 1 (within the limit error) both for control and EAC-treated ghosts. By using tritium-labeled DIDS, 4,4’-diisothiocyanato-2,2’-stilbenedisulfonate ([³H]DIDS), the association rate constant, k₊₁ (M⁻¹s⁻¹), was measured. The mean values of (4.3 ± 0.7) × 10⁵ M⁻¹s⁻¹ for control and (2.7 ± 0.7) × 10⁵ M⁻¹s⁻¹ for EAC-treated ghosts were obtained. The mean values for K_D, evaluated from [³H]DIDS binding measurements, were (37 ± 9) nM and (90 ± 21) nM for control and EAC-modified ghosts, respectively. The results demonstrate that EAC modification of AE1 reduces about 2-fold the affinity of AE1 for DIDS. It is suggested that half of the subunits are modified near the transport site by EAC.     

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.     

Identification of α2 adrenergic receptors in human frontal cortex membranes by binding of [H]RX 821002, the 2-methoxy analog of [H]idazoxan

The antagonist [³H]idazoxan binds with comparable affinity to α₂ adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-³H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300–1200 times lower affinity for the non-stereoselective sites. Their affinity for the α₂ receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [³H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the α₂ receptors (K_D = 2.8 nM than [³H]idazoxan (K_D = 24 nM) and about equal affinity as [³H]rauwolscine (K_D = 3.6 nM).[³H]Rauwolscine binds with comparable affinity to α₂ receptors and to 5-HT_1A receptors, and competition studies indicate that the K_i value of unlabelled RX 821002 for the 5-HT_1A receptors (30 nM) is about one order in magnitude above its K_i value for the α₂ receptors (4.1 nM). Labelling of the 5-HT_1A receptors by [³H]RX 821002 and by [³H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 μM). Under these conditions, specific binding of [³H]RX 821002 to the α₂ receptors represents 84% of total binding (at its K_D), as compared to 77% for [³H]rauwolscine and 20% for [³H]idazoxan.[³H]RX 821002 labels the α₂ receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37°C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [³H]RX821002. About 60% of the α₂ receptors possess high agonist affinity.