3H]yohimbine and [3H]idazoxan bind to different sites on rabbit forebrain and kidney membranes

The binding of the alpha 2-adrenoceptor ligands [3H]yohimbine and [3H]idazoxan to rabbit kidney and forebrain membranes was compared. The maximum number of [3H]yohimbine binding sites was higher than the number of [3H]idazoxan binding sites in forebrain and lower in kidney. Large differences were observed in the ability of noradrenaline, adrenaline, idazoxan, rauwolscine, yohimbine and WY 26392 to displace [3H]yohimbine and [3H]idazoxan from their binding sites. These data suggest that [3H]idazoxan and [3H]yohimbine bind to different sites on rabbit tissue membranes.     

3H]idazoxan and some other alpha 2-adrenergic drugs also bind with high affinity to a nonadrenergic site

We compared the pharmacological properties of the alpha 2-adrenergic radioligand [3H]idazoxan with those of [3H]rauwolscine in rat and [3H]yohimbine in human renal cortical membranes. The density of "specific" [3H]idazoxan binding sites (defined by 100 microM tolazoline) was twice as high as that of [3H]rauwolscine in rat kidney and four times as high as that of [3H]yohimbine in human kidney. A variety of structurally different drugs fully competed for specific [3H]rauwolscine and [3H]yohimbine binding, with affinities appropriate for the interaction with alpha 2-adrenergic receptors. Specific [3H]idazoxan binding, however, was only partially competed for by the catecholamines epinephrine and norepinephrine in both tissues. Thus, [3H]idazoxan labels both alpha 2-adrenergic receptors and a nonadrenergic site. Clonidine, B-HT 920, moxonidine, phentolamine, prazosin, yohimbine, dopamine, and serotonin also could not compete for this site. However, UK 14,304, guanabenz, indanidine, tolazoline, oxymetazoline, and SK&F 104,078 competed for the additional [3H]idazoxan sites with affinities similar to those at alpha 2-adrenergic receptors. [3H]idazoxan binding substantially in excess of [3H]rauwolscine or [3H]yohimbine binding was also found in human platelets, myometrium, and erythroleukemia (HEL) cells but not in three cell lines lacking alpha 2-receptors (MDCK, BC3H1, and Jurkat cells). Although we have been unsuccessful thus far in defining the precise nature of the additional [3H]idazoxan binding sites, we hypothesize that these sites may be closely affiliated with alpha 2-adrenergic receptors but clearly distinct from the catecholamine binding site of the receptor. The results indicate that care must be taken in the use of [3H]idazoxan or drugs that are recognized at its nonadrenergic site when studying alpha 2-adrenergic effects and receptor subtypes.     

External location of sites on pig erythrocyte membranes that bind nitrobenzylthioinosine

Nucleoside transport in erythrocytes of various species is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to high affinity sites associated with nucleoside transport elements of the plasma membrane. The present study examined binding of [3H]NBMPR to unsealed ghosts and to sealed right-side-out vesicles (ROVs) and inside-out vesicles (IOVs) prepared from pig erythrocytes. Kd values for NBMPR dissociation from the ligand-site complex in unsealed ghosts, ROVs and IOVs were similar (1.6-2.4 nM), and Bmax values (mean +/- SD) were, respectively, 22.2 +/- 5.5, 25.8 +/- 6.4, and 37.3 +/- 4.0 molecules/fg of protein, reflecting differences in the protein content of the membrane preparations. When temperatures were decreased from 22 degrees to 4 degrees, NBMPR binding to erythrocyte membrane preparations was reduced in IOVs relative to that in unsealed ghosts and ROVs. At 22 degrees, the association of NBMPR molecules with IOVs was slower than with ROVs and unsealed ghosts, differences that were virtually eliminated by permeabilization of the membrane preparations with saponin. Thus, the binding sites were more accessible to external NBMPR in sealed ROVs and unsealed ghosts than in sealed IOVs, indicating that the NBMPR sites are located on the extracellular aspect of the membrane.     

Identification of alpha 2-adrenoceptors and non-adrenergic idazoxan binding sites in rabbit colon epithelial cells

alpha 2-Adrenoceptors are possibly involved in the regulation of the hydroelectrolytic flux across the digestive mucosa. As no data are available concerning the existence of these receptors in colon epithelial cells, we aimed to investigate the existence of alpha 2-adrenoceptors in this tissue using tritiated antagonists. [3H]Yohimbine and [3H]rauwolscine were not usable to label colonic alpha 2-adrenoceptors because of their very high level of non-specific binding. In contrast, the methoxy derivative of idazoxan, [3H]RX821002, appeared a convenient radioligand for the purpose. [3H]RX821002 bound with high affinity (KD = 6.2 +/- 0.8 nM) to a single population of non-interacting sites (Bmax = 193 +/- 17 fmol/mg protein). The rank order of potency of catecholamine enantiomers and adrenergic drugs to inhibit [3H]RX821002 binding demonstrated that the labelled sites are alpha 2-adrenoceptors and that 53% of the receptors are in a high-affinity state sensitive to GTP + NaCl. [3H]Idazoxan also bound to colocyte membranes, but inhibition by (-)-adrenaline and various imidazoline compounds indicated that this radioligands labels alpha 2-adrenoceptors and non-adrenergic sites. When experiments were performed under binding conditions impeding the interaction of [3H]idazoxan with the alpha 2-adrenoceptors (i.e. in presence of 10(-4) M (-)-adrenaline), the Bmax of non-adrenergic idazoxan binding sites was 97 +/- 8 fmol/mg protein and the KD was 3.5 +/- 0.5 nM. The sites were pharmacologically characterized with various imidazoline and non-imidazoline drugs. In order to study the putative relationship between alpha 2-adrenoceptors and non-adrenergic idazoxan binding sites, the expression of both kinds of sites was investigated along the crypt-to-surface axis. Crypt cells had a higher number of alpha 2-adrenoceptors than surface cells, whereas the number of non-adrenergic idazoxan binding sites remained constant. The results show that (i) alpha 2-adrenoceptors coexist with non-adrenergic idazoxan binding sites in rabbit colocytes; (ii) the number of alpha 2-adrenoceptors is higher in crypt cells than in surface cells and (iii) alpha 2-adrenoceptors and non-adrenergic binding sites are different and unrelated.     

Low affinity binding sites for 1,4-dihydropyridines in mitochondria and in guinea pig ventricular membranes

In this paper, we describe the occurrence of both high and low affinity sites for dihydropyridines in crude membrane preparations from guinea pig ventricular tissue. The physiological significance of the low affinity site (apparent dissociation constant = 76 +/- 9 nM) is not currently known; it has, however, a binding capacity which was 300-1000 times that of the high affinity site and was resistant to heat denaturation. The magnitude of the binding to the low affinity site was affected by both the ionic strength of the medium and by the presence of divalent ions. Both unlabeled nitrendipine and nimodipine inhibited [3H]nitrendipine binding at both sites, but verapamil and diltiazem only affected binding at the high affinity site. We also characterized, both kinetically and by equilibrium binding, a low affinity, heat-stable nitrendipine binding site in purified mitochondria. The Bmax for this site was also dependent on ionic strength. This suggests the possibility that the low affinity site in crude membranes is due to mitochondrial contaminants and hence not directly related to voltage-dependent calcium channels.     

Saturable, high affinity binding of the nucleoside transport inhibitor, nitrobenzylthioinosine, to guinea pig cardiac membranes

The site-specific binding of the potent nucleoside transport inhibitor, [3H]nitrobenzylthioinosine ([3H]NBMPR), to guinea pig cardiac membranes was rapid, reversible and saturable. [3H]NBMPR bound with high affinity to a single class of sites at which the KD was 0.23 +/- 0.07 nM and which had a Bmax of 1700 +/- 290 fmol/mg protein. Several recognized nucleoside transport inhibitors and benzodiazepines inhibited the binding of [3H]NBMPR with an order of potency similar to that observed for the inhibition of the binding of [3H]NBMPR to human erythrocytes and guinea pig synaptosomes.     

Characterization of non-adrenergic [3H]clonidine binding sites in rat stomach: high affinity of imidazolines,guanidines and sigma ligands

We have identified and characterized non-adrenergic [³H]clonidine binding sites in rat stomach. The binding of [³H]clonidine was rapid, reversible, partly specific (as defined by cirazoline 0.1 mmol/l; 68% specific binding at [³H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [³H]clonidine to rat stomach membranes was concentration-dependently inhibited by various imidazolines and guanidines including the sigma site ligand 1,2-di-(2-tolyl)guanidine (DTG), by the butyrophenone derivative haloperidol and by the piperidine derivative (+)-3-PPP[(R)-3-(3-hydroxyphenyl)-N-propylpiperidine]; the latter two compounds are also known to exhibit affinity for sigma sites. In contrast, rauwolscine, histamine, ranitidine and the non-hydrolysable GTP-analogue Gpp(NH)p (5 guanylylimidodiphosphate) did not, or with negligible affinity, inhibit [³H]clonidine binding. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases for a single detectable site) was as follows: cirazoline>idazoxanDTG>(+)-3-PPP> clonidine>guanabenz>haloperidol. This rank order is not compatible with the pharmacological properties of either I₁- or I₂-imidazoline binding sites. However, the ability of haloperidol, (+)-3-PPP and DTG to displace [³H]clonidine (the latter two with high affinity) suggests that the [³H] clonidine binding sites in rat stomach may be related to sigma-like sites.     

Dihydrogenated ergot compounds bind with high affinity to GABAA receptor-associated Cl- ionophore.

The binding of t-[3H]butylbicycloorthobenzoate ([3H]TBOB) to crude synaptosomal membranes of the mouse brain (cerebrum minus cortex) in the presence of dihydroergotoxine, dihydroergosine, dihydroergotamine and gamma-aminobutyric acid (GABA) was studied in vitro. [3H]TBOB binding was inhibited by all drugs used. The rank order of potency was dihydroergotoxine greater than GABA greater than dihydroergosine greater than dihydroergotamine. This suggests that dihydrogenated ergot compounds, especially dihydroergotoxine, possess appreciable binding activity (comparable to that of benzodiazepines and barbiturates) at the GABAA receptor-associated C1- ionophore.     

Binding of radiolabeled high affinity antagonist to leukotriene D4 receptor in guinea pig lung membranes: interconversion of agonist-receptor binding affinity states

The kinetic parameters and the pharmacological specificity of a high affinity leukotriene D4 (LTD4) receptor antagonist, ICI-198615, binding to guinea pig lung membranes were characterized. Binding of [3H]ICI-198615 to the membranes was rapid and displaceable with excess ICI-198615. The specific binding of [3H] ICI-198615 was dependent upon the concentration of membrane protein. Monovalent cations (Na+, Li+, and Cs+), divalent cations (Mg2+, Ca2+, and Mn2+), and guanine nucleotides did not significantly affect the specific binding of [3H]ICI-198615 to guinea pig lung LTD4 receptors. The specific binding of [3H]ICI-198615 to guinea pig lung membranes was saturable and the equilibrium saturation binding was best approximated by a single-site model. The dissociation constant (KD) and the density (Bmax) were 0.08 +/- 0.04 nM and 1030 +/- 180 fmol/mg, respectively. In competition studies, LTD4, stereoisomer (5R,6S)-LTD4, and leukotriene E4 (LTE4) competed with [3H]ICI-198615 binding to specific sites, with a stereoselectivity and a rank order of potency equivalent to those described in [3H]LTD4 binding studies and in functional studies. LTD4 and LTE4 displaced maximally 70 and 40%, respectively, of the [3H]ICI-198615 specific binding component defined by ICI-198615. Several LTD4 receptor antagonists (ICI-198615, WY-48252, WY-49511, FPL-55712, and LY-171883) displaced [3H]ICI-198615 specific binding, with a rank order of potency equivalent to that described in the guinea pig tracheal smooth muscle contraction system. The leukotriene structure-like receptor antagonists, e.g., SK&F 104353 and SK&F 104373, also competed with the [3H]ICI-198615 specific binding, with binding affinities comparable to those expected from the functional studies. However, SK&F 104353 and SK&F 104373 displaced maximally 70% of the specific binding component of [3H]ICI-198615, equivalent to that displaced by LTD4. Guanosine-5'-3-thiotriphosphate (GTP gamma S), EDTA, and Na+ shifted the LTD4 displacement curve to the right, indicating that these agents regulated the binding of LTD4 to the receptor. In the absence of GTP gamma S or cations, the LTD4 displacement curve was heterogeneous. The LTD4 displacement curve was resolved into a higher affinity component (KDH = 0.5 +/- 0.2 nM; percentage of receptor density at high affinity = 24 +/- 3%) and a low affinity component (KDL = 60 +/- 7 nM; percentage of receptor density at low affinity = 76 +/- 3%).(ABSTRACT TRUNCATED AT 400 WORDS)     

3H]Yohimbine binding to human platelet membranes: evidence for high and low affinity binding sites with negative cooperativity

Evidence is presented that [3H]yohimbine binding to human platelet membranes does not follow the simple mass action kinetics. Although [3H]yohimbine binding was saturable and stereospecific, Scatchard analysis of the equilibrium binding data produced a curvilinear plot. Competitive displacement of [3H]yohimbine from the binding sites by unlabeled yohimbine and other alpha 2-antagonists produced shallow inhibition curves. Further, the apparent Hill coefficients of equilibrium binding and competitive displacement data were found to be less than unity. Factors such as binding to nonreceptor sites or to alpha1-adrenoceptors, dopamine receptors, or 5-HT receptors that may explain the curvilinear curve were excluded. The rank order for inhibiting [3H]yohimbine was rauwolscine greater than yohimbine greater than phentolamine greater than clonidine much greater than prazosin, suggesting that the binding sites had the characteristics of alpha2-adrenoreceptors. The affinity of the alpha2-antagonist for the receptor was enhanced by Na+ but not by guanine nucleotide, suggesting that the binding of the antagonist is modulated only by Na+. Graphic analysis of the specific binding data resulted in two components: one with high affinity and low capacity sites, and second with low affinity and high capacity sites. The experiments on dissociation kinetics, however, suggest that the observed deviation of [3H]yohimbine binding from the simple mass action kinetics is most likely due to negative cooperative interactions among alpha2-adrenoceptor sites.     

Specificity of phencyclidine-like drugs and benzomorphan opiates for two high affinity phencyclidine binding sites in guinea pig brain

Recently, the presence of two high affinity binding sites for phencyclidine were described in guinea pig brain, with one site coupled to the glutamate excitatory amino acid receptor, specifically activated by N-methyl-D-aspartate (NMDA) (site 1) and the other site associated with the dopamine (DA) reuptake carrier (site 2). Phencyclidine and its analogs, as well as the benzomorphan opiates, are known to interact with binding sites for phencyclidine. In this study, the equilibrium dissociation constants (Kd) of these compounds for the two binding sites for phencyclidine were determined. Phencyclidine and 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP), an analog of PCP, were essentially non-selective between the two sites and also were the two drugs of the group observed to have the highest affinity for site 2. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine [(+)MK801] was the most selective agent for site 1, while none of the drugs tested showed selectivity for site 2. In humans, phencyclidine produces psychotomimetic effects, while (+)MK801 has been reported to produce minimal, if any, psychotomimetic effects, at doses sufficient to reduce seizures. These clinical observations, in conjunction with the present biochemical binding data, suggest that (+)MK801 may serve as a "marker" for site 1 and that the psychotomimetic effects of phencyclidine might be mediated by site 2.     

Proadifen-sensitive high affinity binding of 3H-alaproclate to liver membranes

3H-Alaproclate, a selective 5-hydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (KD = 3 nM) and large capacity (Bmax about 2 nmol/g liver). This binding was stereoselective since S-(-)-alaproclate was 30 times more potent than the R-(+)-enantiomer to displace the 3H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the 3H-alaproclate binding with the same, high affinity (Ki = 3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5 x 75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced 3H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed.     

Stimulus generalization from cocaine to analogs with high in vitro affinity for dopamine uptake sites

Previous research has shown that phenyltropane derivatives of cocaine are very potent ligands for dopamine transporters in in vitro binding and uptake, and in in vivo binding assays. In the present study, these analogs were tested for their ability to substitute for cocaine in rats trained to discriminate cocaine from saline. Results indicate that these compounds are from 6-13 times more potent than cocaine in producing cocaine-appropriate responding. This provides further evidence in support of the importance of dopamine uptake inhibition for the behavioral effects of cocaine, and suggests utility of these compounds in understanding cocaine abuse.     

125I]SCH 23982, a 'selective' D-1 receptor antagonist, labels with high affinity 5-HT1C sites in pig choroid plexus

[125I]SCH 23982, a ligand reported to be very selective for dopamine D-1 receptors, binds with high affinity to membranes of pig choroid plexus (KD = 186 pM, Bmax = 142 fmol/mg protein). Saturation and competition experiments suggested that [125I]SCH 23982 labels a homogeneous population of sites. The rank order for affinity of agonists, 5-HT greater than or equal to DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) much greater than dopamine greater than fenoldopam, and antagonists, metergoline greater than mesulergine = mianserin greater than SCH 23390 greater than methiothepin greater than ketanserin greater than fluphenazine much greater than (-)-sulpiride greater than (+)-sulpiride, was compatible with labelling of 5-HT1C receptors by [125I]SCH 23982. It also correlates very significantly with [3H]mesulergine binding to pig choroid plexus membranes. The effects of SCH 23390 and its analogues should not be systematically attributed to an interaction with D-1 receptors.     

The effect of two different buffers on the high affinity 3H-ethylketocyclazocine and 3H-SKF10047 binding to guinea pig brain membranes

In vitro mu and delta opioid receptor binding is known to be influenced by ions. High affinity 3H-SKF10047 and 3H-ethylketocyclazocine binding sites are found in brain membranes and postulated to be similar to mu opioid receptor binding. To investigate this postulate, we have studied how the high affinity binding of 3H-SKF10047, 3H-ethylketocyclazocine, a tritiated mu agonist, mu antagonist and delta agonist is altered when the radioreceptor binding assay incubation buffer is changed. The binding of 3H-ethylketocyclazocine and the mu antagonist (3H-naloxone) is highest in isotonic HEPES buffer, while the binding of the mu (3H-dihydromorphine) and delta (3H-D-ala-D-leu-enkephalin) agonist is highest in hypotonic Tris-HCl buffer. 3H-SKF10047 binding is similar in the two buffers. The inhibition of 3H-ethylketocyclazocine, 3H-SKF10047 and tritiated mu and delta opioid ligands by seven unlabeled ligands is then compared in the two buffers. Morphine chloride is a more potent inhibitor of 3H-ethylketocyclazocine binding and tritiated mu ligand in hypotonic Tris-HCl buffer than in isotonic HEPES buffer. The potency of naloxone, nalorphine, SKF10047, D-ala-D-leu-enkephalin, cyclazocine and phencyclidine in inhibiting 3H-ethylketocyclazocine binding is independent of the buffer system. None of the seven unlabelled substances change potency with buffer change when inhibiting the 1.2 nM 3H-SKF10047 binding. In sum our results show that 1 nM 3H-ethylketocyclazocine binding is influenced by buffer change in a manner very similar to mu ligand binding, while the 1.2 nM 3H-SKF10047 binding is only slightly influenced by buffer change and therefore different from mu ligand binding.