Properties of [3H] diazepam binding to rat peritoneal mast cells

Benzodiazepine binding to rat peritoneal mast cells was investigated using [³H] diazepam as the radioactive probe. The specific binding of [³H] diazepam reaches equilibrium within 10–15 min, is saturable and is linear with cell number. Scatchard analysis of equilibrium binding indicates the existence of only one class of binding sites with a K_D = 90 ± 10 nM and B_max of 261 ± 60 fmoles/10⁶ cells. The binding of [³H] diazepam is temperature dependent, the highest amount is bound at 0°C and shows a pH-optimum between pH 6.8 – 7.4. The binding of [³H] diazepam is reversible with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2 ± 0.2}\text{min.}$ Based on the relative potency of clonazepam and Ro5-4864 in displacing the specific [³H] diazepam binding, the binding sites in the mast cell are similar to those in the peripheral tissues like lung, liver, and kidney and are different from those in the brain. These data indicate that the mast cells have benzodiazepine binding sites which are of the peripheral type.     

Calcium alters the properties of [3H]diazepam binding sites in rat brain membranes

• 1.1. [³H]diazepam ([³H]DZ) was used as a ligand to study the effects of Ca²⁺ on benzodiazepine binding to rat brain membranes.
• 2.2. [³H]DZ bound at a single class of binding sites showing K_D and B_max values of 5.4 nM and 852 fmol/mg protein respectively. These values are consistent with previous reports.
• 3.3. Amongst the various divalent cations tested Mg²⁺, Fe²⁺, Cd²⁺, and Sr²⁺ had no significant effect on [³H]DZ binding. Mn²⁺, Ba²⁺, Co²⁺, Ni²⁺ and La²⁺ enhanced radioligand binding, whereas Ca²⁺, Zn²⁺ and Pb²⁺ inhibited [³H]DZ binding to brain freeze-thawed membranes.
• 4.4. The inhibition of [³H]DZ binding by Ca²⁺ was concentration-dependent. 50% inhibition occurred at a Ca²⁺ concentration of 5.6mM. The Hill coefficient for the inhibition was 1.03, displaying noncoperativity. The effect of Ca²⁺ on [³H]DZ binding could be prevented by La³⁺ but was not reversed by EGTA.
• 5.5. A kinetic analysis of Ca²⁺ inhibition of [³H]DZ binding indicates that Ca²⁺ inhibited competitively. Analysis of binding isotherms indicates that both K_D and B_max were altered at the [³H]DZ binding sites. The marked increase in K_D value in the presence of Ca²⁺ (5 mM) can be explained by a drastic increase in the dissociation rate constant.
• 6.6. It was suggested that Ca²⁺ may induce a conformational change in the diazepam binding sites on rat brain membranes. The unchanged Hill coefficient in the presence or absence of Ca²⁺ indicates that a single population of binding sites was labeled by [³H]DZ.
• 7.7. The calmodulin antagonists, trifluoperazine and W-7 were weak inhibitors of [³h]dz binding.

     

Properties of 3H-MPTP binding sites in human blood platelets

Our study demonstrates that 3H-1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (3H-MPTP) specifically binds to platelet membrane sites in humans. This specific, high affinity and saturable binding has properties similar to those of 3H-MPTP binding to rat and monkey brain, with a higher affinity. Deprenyl, a specific inhibitor of MAO type B enzyme, was the most potent drug in displacing 3H-MPTP from platelet binding sites. Platelets are considered a good model for central aminergic neurons and are very rich with MAO enzymatic activity, exclusively of type B. Our findings support previous evidence indicating a correspondence between 3H-MPTP binding sites and MAO-B enzyme. Moreover the presence of 3H-MPTP binding sites on human platelets suggests the use of this peripheral tissue as a simple model to study at least partially the mechanisms of neurotoxic action of MPTP.     

Increased specific binding of [3H]diazepam in rat brain following chronic diazepam administration.

Male rats (175 g) were given 30 mg of diazepam in their food daily for 35 days. The animals became drowsy and ataxic from this high dose of drug. After the 35-day dosing, the rats were killed daily, and specific binding of [3H]diazepam and [3H]flunitrazepam was determined in synaptosomal preparations from these and corresponding control rats. On days 3, 4, 6, and 7 after the treatment period the specific binding and specific binding of [3H]diazepam was double that of the control binding and specific binding of [3H]flunitrazepam was 1.67 times that of control. The data indicate that very high doses of diazepam, given for long periods, cause increased specific binding of radiolabeled ligand to brain subfractions. The possible mechanisms and implications are discussed. When lower doses or shorter dosage regimens are used, increased binding is not observed.     

Multiple binding sites for [3H]clonidine and [3H]WB-4101 in rat brain

Binding of the alpha-adrenergic agonist [3H]clonidine and the alpha-adrenergic antagonist [3H]WB-4101 exhibited multiple binding site characteristics in both rat frontal cortex and cerebellum. Kinetic analysis of the dissociation of both radioligands in rat frontal cortex suggests two high affinity sites for each ligand. Competition of various noradrenergic agonists and antagonists for [3H]WB-4101 binding yielded shallow competition curves, with Hill coefficients ranging from 0.45 to 0.7. This further suggests multiplicity in [3H]WB-4101 binding. In the rat cerebellum, competition of various noradrenergic drugs for [3H]clonidine binding yielded biphasic competition curves. Furthermore Scatchard analysis of [3H]clonidine binding in rat cerebellum showed two high affinity sites with KD = 0.5 nM and 1.9 nM, respectively. Competition of various noradrenergic drugs for [3H]WB-4101 binding in the rat cerebellum yielded biphasic competition curves. Lesioning of the dorsal bundle with 6-hydroxydopamine did not significantly affect the binding of either [3H]clonidine or [3H]WB-4101. These findings for both [3H]clonidine and [3H]WB-4101 binding in rat frontal cortex and cerebellum can be explained by the existence of postsynaptic binding sites for both 3H ligands.     

Biochemical characterization of [3H]tryptamine binding sites from rat brain

Rat brain membranes were treated with different protein modifying reagents, all of which were able to reduce [3H]tryptamine binding. However, inactivation by N-ethylmaleimide and iodoacetamide only was counteracted by coincubation with tryptamine. Thus, the [3H]tryptamine binding molecule is a membrane protein with an essential sulfhydryl group at the binding site. After incubation of digitonin-solubilized membranes with seven different lectins, no precipitation of [3H]tryptamine binding sites was observed. On concanavalin A and wheat germ agglutinin affinity chromatography, no [3H]tryptamine binding activity was found to be specifically bound. Therefore, the [3H]tryptamine binding protein appears to be devoid of lectin binding carbohydrate residues.     

Temperature-sensitive high affinity [3H]tryptamine binding sites in rat brain

The influence of prior incubation on [3H]tryptamine binding was investigated in rat brain synaptic plasma membranes. A 55 min preincubation of the membranes at 37 degrees C induced an approx. 2.4-fold increase in the specific binding of [3H]ligand to the subsequently washed preparations and this phenomenon was quite temperature-dependent. On the other hand, the proportion of nonspecific binding sites was significantly decreased by 70% of the original sites within 20 min of the start of preincubation. Pargyline, ascorbic acid, EGTA, metal ions (Ca2+, Mg2+, Na+) and guanine nucleotides, included in the preincubation buffer, were all inactive on the stimulation of [3H]tryptamine binding, while the pretreatment of membranes with glutaraldehyde antagonized the augmentation of this binding. Furthermore, it was revealed that the Scatchard plot of the [3H]tryptamine binding preincubated at 0 degree C conformed to a straight line (KD = 33.1 nM, Bmax = 543 fmoles/mg protein), whereas a curvilinear Scatchard plot was obtained at 37 degrees C preincubation. Nonlinear regression analysis of the latter resulted in apparent KD (nM) & Bmax (fmoles/mg protein) values of 0.45 & 102.7 and 33.7 & 603.4 for the high and low affinity sites, respectively. All these observations lead to the inference that the preincubation-induced increase in [3H]tryptamine binding (i.e., nearly high affinity proportion of sites) may occur as a result of temperature-sensitive interconvertible conformational changes.     

3H-spiroperidol binding sites in blood platelets

3H-spiroperidol, an antagonist of dopamine receptors in brain (striatum), was found to bind to human and rat platelet membrane preparations. The binding was rapid, reversible, saturable and specific. Unlabelled haloperidol displaced the specifically bound 3H-spiroperidol. Binding equilibrium was attained in 15 min at pH 7.4 and 37 degrees C. Scatchard analysis of 3H-spiroperidol binding revealed a single population of binding site with Kd of 7.6 nM in rat platelet membrane and Kd of 15 nM in human platelet membrane. Unlabelled 5-hydroxytryptamine produced no significant effect on 3H-spiroperidol binding to rat or human blood platelet membranes in the presence or absence of haloperidol. Some dopaminergic agents, known to inhibit spiroperidol binding in corpus striatum, also inhibited the same in rat and human blood platelet membranes under in vitro conditions. This study suggests the presence of specific 3H-spiroperidol binding sites in blood platelets.     

Demonstration of specific "high affinity" binding sites for [3H] imipramine on human platelets

High affinity and saturable binding sites for [³H] imipramine have been demonstrated on human platelet membranes. These binding sites appear to be specific for tricyclic antidepressants and their pharmacologically-active metabolites. In contrast, inactive tricyclic compounds such as the parent iminodibenzyl and iminostilbenes do not inhibit [³H] imipramine binding. The binding of [³H] imipramine to human platelets is of high affinity $\text{(}\text{Kd}\text{? 1.4}\text{nM}\text{)}$, saturable $\text{(}\text{Bmax}\text{? 625}\text{fmols/mg prot}\text{)}$, and sensitive to proteolytic degradation. The effects of various drugs and neurotransmitter agonists and antagonists suggests that these binding sites are pharmacologically distinct from the previously reported binding of tricyclic antidepressants to alpha-adrenergic, muscarinic-cholinergic, and histaminergic receptors. The binding characteristics of [³H] imipramine to platelets is similar to that in rat and human brain and may thus serve as a useful model in elucidating the pharmacological and physiological significance of these binding sites.     

Norharman inhibition of [3H]diazepam binding in mouse brain

Norharman competitively inhibits specific binding of [³H]-diazepam in mouse brain homogenates. $\text{In vivo}$ this β-carboline produces a striking rigid catatonic-like appearance which is abolished by diazepam. It also causes a rapid tremor but has little anticonvulsant effect. Measurement of $\text{in vivo}$ concentrations and receptor occupancy demonstrate that these biological effects occur at doses which occupy a large proportion of benzo-diazepine receptors. It may represent a ligand of the benzo-diazepine receptors whose effects are opposite those of diazepam.     

In situ molecular weight determination of rat brain [3H]phenytoin binding sites

Using irradiation inactivation analysis of specific [3H]phenytoin binding to rat brain we have demonstrated that there are two different binding sites involved, with molecular weights of 73238 +/- 1535 (higher affinity site) and 108121 +/- 6935 (lower affinity site).     

Effects of trypsin on binding of [3H]epinephrine and [3H]-dihydroergocryptine to rat liver plasma membranes. Evidence for interconversion of binding sites

Treatment of liver plasma membranes with trypsin at low concentrations (1 to 2 microgram/mg of protein) caused at 3- to 4-fold increase in alpha-specific [3H]epinephrine binding. The change was due to an increase in the number of high affinity binding sites, with no change in the dissociation constant. With increasing trypsin concentrations, the dissociation constant was decreased and there was a progressive loss of binding. Elastase, papain, and thermolysin caused similar effects, whereas the thrombin, leucine aminopeptidase, phospholipase A2, phospholipase C, phospholipase D, and detergents did not cause an increase in [EH]epinephrine binding. The increase in epinephrine high affinity binding sites was correlated with a loss of high affinity [3H]-dihydroergocryptine binding sites which also bind [3H]epinephrine with low affinity (El-Refai, M. F., Blackmore, P. F., and Exton, J. H. (1979) J. Biol. Chem. 254, 4375-4386). Incubation of membranes with the alpha blockers dihydroergocryptine (50 nM) and phenoxybenzamine (20 nM) prior to protease treatment diminished the increase in [3H]epinephrine binding induced by trypsin (1.5 microgram/mg). The concentration dependence and time course of trypsin actions on 70 nM [3H]epinephrine binding and 10 nM [3H]dihydroergocryptine binding are consistent with a trypsin-mediated conversion of low affinity epinephrine binding sites to high affinity epinephrine binding sites.     

3H] diazepam binding to human granulocytes

[3H]-diazepam binds to sites on human granulocyte membranes, with little or no binding to platelets or lymphocytes. These [3H]-diazepam binding sites are of the peripheral type, being strongly inhibited by R05-4864 (Ki = 6.23nM) but only weakly by clonazepam (Ki = 14 microM). Binding of [3H] diazepam at 0 degree is saturable, specific and stereoselective. Scatchard analysis indicates a single class of sites with Bmax of 109 +/- 17f moles per mg of protein and KD of 3.07 +/- 0.53nM. Hill plots of saturation experiments gave straight lines with a mean Hill coefficient of 1.03 +/- 0.014. Binding is time dependent and reversible and it varies linearly with granulocyte protein concentration over the range 0.025-0.300 mg of protein.     

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

Specific binding sites for 5-hydroxytryptamine on rat blood platelets.

5-Hydroxytryptamine changes the shape of rat blood platelets by combination with a cinanserin-sensitive receptor which is not associated with the active uptake of 5-hydroxytryptamine. Binding of 5-hydroxy[3H]tryptamine to platelets at 4 degrees C demonstrates the presence of three saturable sites, and the highest-affinity site is apparently this 5-hydroxytryptamine receptor.     

[3H]Serotonin binding sites in goldfish retinal membranes

Serotonin (5HT) binding sites were studied in goldfish retinal membranes by radioligand experiments. The binding site of [³H]5HT was sensitive to pre-treatment of the membranes at 40° or 60° C. 5HT and 5-methoxy-N,N-dimethyltryptamine were the best inhibitors of [³H]5HT binding to retinal membranes. The 5HT₂ agonist, 1-(-naphtyl)piperazine, was also a potent inhibitor, however, (+)-1-2,5-dimethoxy-4-iodopheny1-2-aminopropane was less efficient. The catecholaminergic agents haloperidol and clonidine did not display an important inhibition. Propranolol, also reported as 5HT_1B antagonist, was a relatively potent blocker. Monoamine uptake blockers did not show potent inhibition. The GTP analog, GppNHp, inhibited the binding. The iterative analysis of saturation curves revealed two classes of binding sites, a high affinity component (B_max 2.45 pmol/mg of protein, k_d 6.86 nM), and a low affinity component (B_max 53.46 pmol/mg of protein, K_d 232.07 nM). Analysis of the association and dissociation kinetics suggested a binding site (K_d 2 nM). The semilogarithmic plot of the dissociation kinetics gave curves concave to the upper side. The selectivity of the binding and the inhibition by GppNHp suggest the existance of 5HT₁ receptors in goldfish retina. The low affinity interaction probably represents the transporter of 5HT or a suptype of receptor expressed in glial cells.Abbreviations used B _max maximum binding capacity - CPP, 1 (3 chlorophenyl)piperazine - CLN clonidine - DMI desimipramine - DMT 5-methoxy-N,N-dimethyltryptamine - DOI (+)-1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane - DPAT (+)-8-hydroxy-2-(D1-N-propylamino)tetralin - GppNHp 5-guanylylimidodiphosphate - HAL haloperidol - 5HT serotonin - IC₅₀ concentration of drug producing 50% inhibition of binding - IMI imioramine - K_d equilibrium dissociation constant - MIAN mianserin - NOM nomifensin - NP 1-(1-napthyl)piperazine - PRP propranolol In memory of Dr. Boris Druian who died on Dec. 24, 1991.     

Detailed analysis of heterogeneity of [3H]naloxone binding sites in rat brain synaptosomes

The experiments reported in this paper address the question of heterogeneity of [³H]naloxone binding sites in rat brainstem synaptosomal preparations at 23°C in the presence of 100 mM sodium chloride. Kinetic analysis in the presence of 0.4, 4 and 10 nM [³H]naloxone gave pseudo-first order association rate of 0.9±0.04, 1.23±0.08 and 1.06±0.08 min^–1, respectively. The dissociation of a 1 nM [³H]naloxone receptor complex was biphasic with dissociation rate constants of 1.8 and 0.4 min^–1. On the other hand, dissociation of 10 nM [³H]naloxone was monophasic with ak _d of 1.1 min^–1. Two subpopulations of binding sites were also observed by steady state binding studies, with Kd values of 0.5 and 3.4 nM and a ratio of high to low affinity sites of 1:9. Heterogeneity of [³H]naloxone binding sites could be seen by displacement studies performed with opioid eptides and alkaloids. We suggest that our data best fits a model with two independent naloxone binding sites wherein either one or both undergo a multi-step interaction with ligand.     

Characterization of [H]glutamate binding sites on frozen sections from rat adrenal

Some biochemical characteristics of [³H]glutamate (Glu) binding sites on frozen sections from the rat adrenal glands were studied. Adrenal frozen sections exhibited stereo-selective, saturable and temperature-dependent binding of [³H]Glu. An agonist for one of the subclasses of central Glu receptors, quisqualic acid (QA), elicited a significant inhibition of the binding, whereas neither N-methyl- -aspartic acid nor kainic acid, agonists for other subclasses of the receptors, had such a significant effect on the binding at the concentration range similar to QA. In vitro addition of sodium acetate (100 mM) resulted in a significant inhibition of [³H]Glu binding to frozen sections of the rat adrenal glands. It thus appears that there exist QA-sensitive binding sites of [³H]Glu in the rat adrenal glands which exhibit pharmacological characteristics distinctly different from those in the brain.