Endogenous regulators of benzodiazepine recognition sites

The method used to prepare crude synaptic membranes (CSMs) from rat brain affects the results obtained for the binding characteristics of ³H-diazepam and the GABA-induced stimulation of ³H-diazepam to CSM. In freshly prepared membranes (rich in GABA and other endogenous inhibitory factors), the K_D for ³H-diazepam is approximately 10 nM and the threshold dose of GABA needed to stimulate this binding is approximately 10^?5M. Removal of GABA resulted in an increase in the K_D values for ³H-diazepam binding. In contrast removal of endogenous inhibitory factors (by treatment of the membranes with Triton X-100) resulted in a decrease of the K_D values. In the Tritron X-100 treated membranes the threshold dose of GABA (10^?8M) required to stimulate ³H-diazepam binding is in the range of the high affinity component of ³H-GABA binding. Addition of crude preparations of endogenous inhibitor to these membranes increased the K_D of ³H-diazepam and inhibited the GABA-induced stimulation of ³H-diazepam binding.     

GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors

The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [³H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 M) or THIP plus the antagonist bicuculline methobromide (150 M of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA with a binding constant (K _D) of 7.9±0.4 nM and aB _max of 3.42±0.08 pmol×mg^–1 protein. Membranes from cells cultured in the presence of THIP had two binding sites for GABA withK _D-values of 6.8±0.9 nM and 476±311 nM, respectively. The correspondingB _max values were 4.41±0.42 pmol×mg^–1 and 5.81±1.20 pmol×mg^–1. The effect of culturing the cells in THIP was antagonized by the simultaneous presence of bicuculline in the culture media, i.e. no significant low-affinity binding for GABA was found on the membranes from granule cells cultured in both THIP and bicuculline. TheK _D value (14.3±1.4 nM) for the high affinity binding site was, however, slightly increased compared to the non-treated cells. These findings suggest that the ability of THIP to induce formation of low-affinity GABA receptors is mediated by preexisting high-affinity GABA-receptors on the granule cells.     

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.     

[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.     

Changes in mouse brain diazepam receptor binding after phenobarbital administration

Specific ³H-diazepam binding was measured $\text{in vitro}$ in adult mouse (strain, Crl=CD-1) brain after four days of an inductive dose of phenobarbital pretreatment (i.p.). Sexual dimorphism was observed in ³H-diazepam brain binding, female mice had significantly higher benzodiazepine binding than males without any differences in apparent affinity constants (K_D). Phenobarbital pretreatment caused a significant decrease in the maximal number of binding sites (Bmax) as well as in dissociation rate constants in both sexes.     

Reduced diazepam binding following chronic benzodiazepine treatment.

Specific ³H-diazepam binding was measured in rat cortex after 7–10 days of twice daily injection of a large dose of flurazepam. Compared to cortex taken from saline treated controls, there was a statistically significant decrease of about 15% in the maximum binding capacity (B_max). There was also a change in the dissociation constant (K_D) from 5.27 to 8.80 nM. Repeated washing of the tissue sample before the binding assay showed that the B_max was truly decreased in the treated animals, but the change in K_D was probably an artifact due to residual flurazepam remaining in the tissue that interfered with the binding assay. It is concluded that chronic benzodiazepine treatment caused an apparent decrease in the number of specific binding sites.     

Peripheral dopamine receptor identification: Properties of a specific dopamine receptor in the rat adrenal zona glomerulosa

Specific dopaminergic receptors were found in the rat adrenal zona glomerulosa. Specific binding as defined by the difference in [³H]-spiroperidol binding in the presence or absence of excess dopamine was saturable and of high affinity. Stereospecificity of binding to the dopaminergic receptor was demonstrated by the fact that (+)-butaclamol was 300-fold more active at displacing [³H]-spiroperidol from the binding site than (?)-butaclamol. A Scatchard analysis of the data revealed a K_D = 6.9 nM and a B_max = 173 pmol/gm for the binding of [³H]-spiroperidol to adrenal capsular homogenate binding site. Characteristics of this receptor place it in the recently defined D2 dopamine receptor subclass.     

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 [3H]phencyclidine to membranes from housefly thoracic muscles

The binding of [³H]phencyclidine ([³H]PCP) to a preparation of housefly thoracic muscle membranes was studied. Specific [³H]PCP binding saturated with both time and at concentrations of the radiolabeled ligand greater than 20 nM. One binding site with a K_D of 10.7 nM and a B_max of 3.9 pmol/mg protein was observed. Specific [³H]PCP binding was also readily reversible with a half-time of dissociation (t_1/2) of 13.8 min and varied proportionately with tissue concentration. Of the drugs tested, specific [³H]PCP binding was inhibited by PCP analogs, antipsychotics, antidepressants, Ca²⁺ channel antagonists, and K⁺ channel blockers. [³H]PCP binding was unaffected by addition of carbamylcholine or L-glutamate in absence or presence of ATP, GTP, cAMP, or cGMP. Though the identity of the [³H]PCP binding protein in housefly muscle membranes is still unclear, it is more likely to be an ionic channel such as K⁺ or Ca²⁺ channels than a neurotransmitter receptor.     

[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.     

Specific binding of phorbol ester tumor promoters to mouse skin

The tumor promoter 20-³H-phorbol 12,13-dibutyrate bound in a specific manner to particulate preparations from both whole mouse skin and mouse epidermis. The binding, which was comparable in both whole skin and epidermal preparations, occurred rapidly, was reversible upon addition of non-radioactive ligand and showed high affinity (K_D = 2.4 × 10^?8 M). The potencies of phorbol esters for inhibiting binding of ³H-PDBu corresponded to their biological and tumor-promoting activities: phorbol 12-myristate 13-acetate, K_I = 0.74 nM; phorbol 12,13-didecanoate, K_I = 16 nM; phorbol 12,13-dibenzoate, K_I = 82 nM; mezerein, K_I = 98 nM; phorbol 12,13-diacetate, K_I = 3 μM; phorbol 12,13,20-triacetate, K_I = 5.6 μM; phorbol 13-acetate, K_I = 64 μM. The biologically inactive derivatives phorbol (0.88 mM) and 4α-phorbol 12,13-didecanoate (15 μM) did not inhibit binding. Likewise, ³H-PDBu binding was only weakly inhibited by phorbol-related diterpenes which are highly inflammatory but nonpromoting. These structure-activity relationships suggest that the ³H-PDBu binding activity mediates phorbol ester tumor promotion. ³H-PDBu binding was not inhibited by the nonphorbol promoters examined. Similarly, it was not blocked by compounds which antagonize (dexamethasone acetate, 2 μM; retinoic acid, 10 μM) or mimic (epidermal growth factor, 100 ng/ml; melittin, 25 μg/ml; PGE₂, 1 μM) some of the effects of the phorbol esters in vivo or in vitro.     

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.     

Interactions of Heparin with Alpha-2-Adrenoceptors

Abstract

The interactions of the anticoagulant Heparin with the alpha-2-adrenoceptor in rat brain cortex membranes were investigated. Binding experiments with ³H-Clonidine were performed in both the absence and presence of Heparin. 1 uM Na-Heparin caused a significant decrease in the maximal number of binding sites (B_max) from 129.4 fmol/mg protein to 93.7 fmol/mg protein with an associated decrease in affinity (K_D = 0.79 pM vs. K_D= 1.53 pM) of these binding sites. Addition of Na⁺-Heparin to ³H-Clonidine (3.1 nM) labelled membranes inhibited 50% of specific ³H-Clonidine binding (IC₅₀) at a concentration of 0.95 uM. Based on our findings we conclude that the simultaneous long term administration of Na-Heparin and the antihypertensive agonist Clonidine should be regarded under consideration of the inhibitory effect of Na-Heparin to the alpha-2-adrenoceptors.     

Etomidate stereospecifically stimulates forebrain,but not cerebellar, 3H-diazepam binding

³H-Diazepam binding to a total particulate fraction of rat forebrain is enhanced by (+)-etomidate and GABA, but not by (?)-etomidate. The enhancement of ³H-diazepam binding by (+)-etomidate was due to a two-fold increase in binding affinity, the maximal number of sites remained unchanged. The degree of stimulation with (+)-etomidate was higher than that obtained with GABA. THIP did not stimulate ³H-diazepam binding to forebrain, and did not reverse the enhanced binding seen with (+)-etomidate or GABA. In a synaptosomal membrane preparation of rat cerebellum, unlike (+)-etomidate, GABA and muscimol produced a marked stimulation of ³H-diazepam binding. (+)-Etomidate did not inhibit ³-muscimol binding to GABA receptors, nor did it activate or inhibit other $\text{in vitro}$ receptor binding assays. The effects of (+)-etomidate on the benzodiazepine binding are different from those of gabamimetic drugs. It is proposed that like barbiturates, (+)-etomidate may affect benzodiazepine binding by interaction with the chloride ionophore which is coupled to the GABA-receptor.     

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.     

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.     

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.     

Elevation of [3H]cimetidine binding by CuCl2 in brain membranes of rats

Influences of dithiothreitol (DTT), p-chloromercuriphenyl sulfonate (PCMPS) and ascorbate on CuCl₂-induced elevation of [³H]cimetidine binding were investigated in brain membranes of rats. CuCl₂ (10–500 μM) elevated specific [³H]cimetidine binding in a concentration-dependent manner. There were two types of [³H]cimetidine binding in the presence of 50 μM CuCl₂: high affinity binding with K_d = 1.97 nM and low affinity with K_d = 21.6 nM. PCMPS (10 and 100 μM) reduced the binding in both media with and without CuCl₂. DTT (1–30 μM) or ascorbate (0.1 and 1.0 mM) markedly elevated the binding in the presence of CuCl₂ but showed no effect and ascorbate rather inhibited the binding in the absence of CuCl₂. DTT (0.1 mM) diminished the binding in the presence and absence of CuCl₂. CuCl₂ (50 μM) significantly (P < 0.01) increased the IC₅₀ of histamine for [³H]cimetidine binding and the effect was greater than that from 100 μM GTP. It is suggested that sulfhydryl groups sensitive to PCMPS could interact with Cu²⁺ and thus be involved in an elevation of cimetidine binding. Cu²⁺ seems to regulate affinity of agonist binding for cimetidine binding sites presumably by acting on cimetidine binding sites and/or GTP binding regulatory proteins.     

Binding of [3H]batrachotoxinin A-20-α-benzoate to a high affinity site associated with house fly head membranes

1. [³H]Batrachotoxinin A-20-α-benzoate (BTX-B), a radioligand that labels the alkaloid activator recognition site of the voltage-sensitive sodium channel, was bound specifically to high affinity, saturable sites in a subcellular preparation from house fly (Musca domestica L.) heads that was shown previously to contain binding sites for other sodium channel-directed ligands.2. Specific binding of [³H]BTX-B was observed in the presence of 140 mM sodium or potassium and was inhibited by choline ion.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom stimulated the specific binding of [³H]BTX-B four-fold, increasing the proportion of specific binding of 10 nM [³H]BTX-B from less than 15% to 40%. Equilibrium dissociation studies in the presence of scorpion venom gave an equilibrium dissociation constant (K_D) for [³H]BTX-B of 80 nM and a maximal binding capacity (B_max) of 1.5 pmol/mg protein.4. Parallel experiments in the absence of venom gave a K_D value of 140 nM and a B_max of 1.3 pmol/mg protein, indicating that scorpion venom stimulated [³H]BTX-B binding by increasing the affinity of this site approximately two-fold.5. The specific binding of [³H]BTX-B was inhibited by the sodium channel activators aconitine and batrachotoxin and, to a lesser extent, by the anticonvulsant diphenylhydantoin. However, several other sodium channel-directed neurotoxins known to exert allosteric effects on the binding of [³H]BTX-B to mammalian brain preparations did not affect the binding of [³H]BTX-B to house fly head membranes.6. These studies provide evidence for a high affinity binding site in house fly head membrane preparations that exhibits properties expected of the activator recognition site of the voltage-sensitive sodium channel but does not respond to several compounds known to modify allosterically the binding of [³H]BTX-B to sodium channels in mammalian brain.