[3H]Spiroxatrine labels a serotonin1A-like site in the rat hippocampus

[³H]Spiroxatrine was examined as a potential ligand for the labeling of 5-HT_1A sites in the rat hippocampus. Analysis of the binding of [³H]spiroxatrine in the absence and presence of varying concentrations of three monoamine neurotransmitters revealed that serotonin (5-HT) had high affinity (IC₅₀= 20.7 nM for the [³H]spiroxatrine binding sites, consistent with the labeling of 5-HT₁ sites, while dopamine and norepinephrine had very low affinity (IC₅₀=57600 nM and >10⁻⁴ M respectively). Saturation studies of the binding of [³H]spiroxatrine revealed a single population of sites with a K_d=2.21 nM. Further pharmacologic characterization with the 5-HT_1A ligands 8-hydroxy-2-(di-n-propylamino) tetralin, ipsapirone, and WB4101 and the butyrophenone compounds spiperone and haloperidol gave results that were consistent with [³H]spiroxatrine labeling 5-HT_1A sites. This ligand produced stable, reproducible binding with a good ratio of specific to nonspecific binding. The binding of [³H]spiroxatrine was sensitive to GTP, suggesting that this ligand may act as an agonist. This was supported by the finding that spiroxatrine inhibits forskolin-stimulated adenylate cyclase activity (a proposed 5-HT_1A receptor model) in the rat hippocampus. Since [³H]spiroxatrine is structurally distinct from other currently available radioligands for the 5-HT_1A site, it should provide new information about the properties of this putative serotonergic receptor.     

Autoradiographic characterization of binding sites for [3H]NE-100 in guinea pig brain

The receptor binding specificity and neuroanatomical distribution of [³H]NE-100 (N, N- dipropyl-2- [4- methoxy-3- (2- phenylethoxy) phenyl] ethylamine monohydrochloride)-labeled sigma receptor in guinea pig brain were examined using quantitative autoradiography. NE-100 potently inhibited [³H]NE-100 binding to slide-mounted sections of guinea pig brain with the IC₅₀ value of 1.09 nM, therefore, NE-100 apparently has high affinity binding sites. Competition studies, under conditions similar to those used to visualize the receptor, yielded the following rank order of potency: NE-100 > haloperidol > DuP734 > (+)pentazocine ⪢ (−)pentazocine. Non-sigma ligands such as phencyclidine (PCP), MK-801 and (−)sulpiride had negligible affinities for [³H]NE-100 binding sites. High densities of [³H]NE-100 binding sites displaceable by haloperidol were present in the granule layer of the cerebellum, the cingulate cortex, the CA3 region of the hippocampus, the hypothalamus and the pons. The distribution of [³H]NE-100 binding sites was consistent with that of [³H](+)pentazocine, a sigma₁ ligand. These sigma sites may possibly be related to various aspects of schizophrenia.     

[3H]Tyramine binding: A comparison with neuronal [3H]-dopamine uptake and [3H]mazindol binding processes

[³H]Spiperone ([³H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [³H]tyramine ([³H]PTA), thus indicating that [³H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [³H]PTA binding and [³H]DA uptake processes were markedly decreased, with no effect on [³H]mazindol ([³H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [³H]PTA binding, did not affect the [³H]MAZ binding process. This further supported the suggestion that while [³H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [³H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras     

Allosteric modulation of the [3H]quinuclidinyl benzylate binding to M-cholinoreceptors in rat cerebral cortex by adrenotropic agents

The allosteric effects of adrenotropic drugs and the membranotropic agent cocaine on the kinetics of [³H]quinuclidinyl benzylate ([³H]QNB) binding to muscarine cholinoceptors of synaptosomal membranes of rat cerebral cortex were studied. In control, the best results were obtained for the model that assumes the existence of two receptor pools (with high and low affinity) with calculated parameters of the activity (K _d), amount (B _max), and mono- to dimer receptors ratio (n). For the high-affinity receptors these parameters were K _d1 = 0.18 ± 0.08 nM, B _m1 = 221.2 ± 56.7 fmol/mg protein, n ₁ = 2, and for low-affinity receptors, K _d2 = 1.33 ± 0.11 nM, B _m2 = 748.7 ± 53.3 fmol/mg protein, n ₂ = 2. Allosteric modulation of the activity of specific neurotransmitter receptors can be accomplished by changing the receptor affinity and amount as well as the proportion of mono- and dimer receptors. Under control conditions, muscarine receptors exist as dimers. In the presence of α-adrenoreceptor agonist noradrenaline and β-adrenoreceptor antagonist propranolol, only one pool of the dimer muscarine receptors remains. The number of binding sites for noradrenaline and propranolol decreases approximately by 40% and 20%, respectively. The agonist of α₂-adrenoreceptors clonidine, the antagonist of α₂-adrenoreceptors yohimbine, and a membranotropic agent cocaine change the ligand binding so that only one receptor pool remains but some of the dimer receptors become monomeric (1 < n < 2). The amount of binding sites reduces by 20%, 25%, and 45% for clonidine, yohimbine, and cocaine, respectively.     

Solubilization of quisqualate-sensitive L-[3H]glutamate binding sites from guinea pig brain membranes using a zwitterionic detergent

L-[3H]Glutamate binding sites were solubilized with a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) plus ammonium thiocyanate from guinea pig synaptosomal membranes. The binding of L-[3H]glutamate to the solubilized binding sites was saturable and reversible. Scatchard analysis suggested the existence of two different classes of binding sites with KDs of 63.8 and 644 nM. The L-[3H]glutamate binding was displaced by excitatory amino acids with such an order of potency that L-glutamate much greater than D-glutamate congruent to L-aspartate greater than D-aspartate. Quisqualate effectively displaced the glutamate binding in biphasic manner. L-Glutamic acid diethyl ester, the quisqualate receptor antagonist, also showed a moderate displacing ability. Other neuroactive amino acid analogues displaced the glutamate binding only weakly, except for L- and D-homocysteic acids which had moderate potency. It is very likely from these results that the glutamate binding sites solubilized in this study are relevant to the physiological glutamate receptors especially of quisqualate-type.     

Studies of the biogenic amine transporters. 1. Dopamine reuptake blockers inhibit [3H]mazindol binding to the dopamine transporter by a competitive mechanism: Preliminary evidence for different binding domains

The present study addressed the hypothesis that the DA transporter ligand, [³H]mazindol, labels multiple sites/states associated with the dopamine (DA) transporter in striatal membranes. Incubations with [³H]mazindol proceeded for 18–24 hr at 4C in 55.2 mM sodium phosphate buffer, pH 7.4, with a protease inhibitor cocktail. In order to obtain data suitable for quantitative curve fitting, it was necessary to repurify the [³H]mazindol by HPLC before a series of experiments. Under these conditions, we observed greater than 80% specific binding. The method of binding surface analysis was used to characterize the interaction of GBR12935, BTCP, mazindol, and CFT with binding site/sites labeled by [³H]mazindol. A one site model fit the data as well as the two site model: Bmax=16911 fmol/mg protein, Kd of [³H]mazindol=75 nM, Ki of GBR12935 =8.1 nM, Ki of CFT=50 nM and Ki of BTCP=44 nM. The inhibitory mechanism (competitive or noncompetitive) of several drugs (GBR12935, CFT, BTCP, cocaine, cis-flupentixol, nomifensine, WIN35,065-2, bupropion, PCP, and benztropine) was determined. All drugs inhibited [³H]mazindol binding by a competitive mechanism. Although the ligand-selectivity of the [³H]mazindol binding site indicates that it is the uptake inhibitor recognition site of the classic DA transporter, the quantitative differences among the ligand-selectivities of different radioligands for the same site suggest that each radioligand labels different overlapping domains of the DA uptake inhibitor recognition site. It is likely that development of domain-selective drugs may further our under-standing of the DA transporter.     

High affinity stereospecific binding of [3H] cocaine in striatum and its relationship to the dopamine transporter

A high affinity (KD 35 nM) binding site for [3H]cocaine is detected in rat brain striatum present at 2-3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [3H]dopamine ([3H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [3H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [3H]cocaine binding stereospecifically, but with lower potency (IC50 approximately equal to 1 microM) than does cocaine. It is suggested that the DA transporter in striatum is the putative "cocaine receptor." Binding of [3H]cocaine, measured in 10 mM Na2HPO4-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na+ and K+ and by biogenic amines. DA and Na+ reduce the affinity of the putative "cocaine receptor" for [3H]cocaine without changing the Bmax, suggesting that inhibition may be competitive. However, TRIS reduces [3H]cocaine binding noncompetitively while Na+ potentiates it in TRIS buffer. Binding of [3H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [3H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na+ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.     

Temperature dependence and GABA modulation of [3H]triazolam binding in the rat brain

The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Our major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of [3H]TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (Kd = 0.27 +/- 08 nM at 0 degree C; Kd = 1.96 +/- 0.85 nM at 37 degrees C) while the Bmax values remained unchanged (1220 +/- 176 fmoles/mg protein at 0 degree C and 1160 +/- 383 fmoles/mg protein at 37 degrees C). Saturation studies of [3H]TZ binding in the presence or absence of GABA (100 microM) showed a GABA-shift. At 0 degrees C the Kd values were (Kd = 0.24 +/- 0.03 nM/-GABA; Kd = 0.16 +/- 0.04/+GABA) and at 37 degrees C the Kd values were (Kd = 1.84 +/- 0.44 nM/-GABA; Kd = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, our findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists.     

3H]U69,593 binding in guinea-pig brain: comparison with [3H]ethylketazocine binding at the kappa-opioid sites

[3H]U69,593 and [3H]ethylketazocine (mu + delta suppressed) binding was measured in homogenates of guinea-pig brain. Both ligands bind with high affinity to a single class of opioid sites. The relative equilibrium dissociation constant (KD) for [3H]U69,593 is 1.15 nM, while [3H]ethylketazocine has a KD value of 0.33 nM. Their respective maximum binding capacities are 4.49 and 4.48 pmol/g of wet tissue. Various mu-selective, delta-selective, kappa-selective, and nonselective opioids were tested in competition studies against the binding of [3H]U69,593 or [3H]ethylketazocine (in the presence of mu- and delta-blockers) to measure their relative affinity. [D-Ala2, MePhe4,Gly5-ol]enkephalin (mu-selective) has low affinity (600-3000 nM) and [D-Pen2,D-Pen5]enkephalin and [D-Ser2, Leu5, Thr6]enkephalin (delta-selective) have very low affinities (greater than 20,000 nM) at the sites labelled with [3H]U69,593 or [3H]ethylketazocine. On the other hand, unlabelled U69,593, U50,488H, and tifluadom (all three kappa-selective substances) display high affinity (1-5 nM) at those sites. Nonselective opioids, such as bremazocine, levorphanol, and ethylketazocine show similar affinities at the sites labelled with [3H]U69,593 and at the sites labelled with [3H]ethylketazocine. These data indicate that [3H]U69,593 is a selective high-affinity ligand for the same sites that are labelled with [3H]ethylketazocine (in the presence of mu- and delta-blockers) and that these are kappa-sites.     

Ropizine concurrently enhances and inhibits [3H]dextromethorphan binding to different structures of the guinea pig brain: autoradiographic evidence for multiple binding sites

Ropizine (10 microM) produces a simultaneous enhancement and inhibition of [3H]dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [3H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhanced [3H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)-pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects of ropizine are due, at least in part, to the effects of ropizine on two different types of [3H]DM binding sites. However, this study does not rule out that common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.     

Repizine concurrently enhances and inhibits [3H] dextromethorphan binding to different structures of the guinea pig brain: Autoradiographic evidence for multiple binding sites

Ropizine (10 μM) produces a simultaneous enhancement and inhibition of [³H]dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [³H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhanced [³H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)-pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects of ropizine are due, at least in part, to the effects of ropizine on two different types of [³H]DM binding sites. However, this study does not rule out thet the common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.     

Characterization of a dopamine-sensitive [3H]LSD binding site in honeybee (Apis mellifera) brain

[³H]Lysergic acid diethylamide ([³H]LSD) binds on membrane homogenate of honeybee brain to both a dopamine-sensitive site (D-site) and a serotonin-sensitive site (S-site). Under suitable conditions the properties of the two sites can be studied separately. Specific binding of [³H]LSD to both the D-site and the S-site has high affinity and is saturable. The mean equilibrium dissociation constants (K_D) were 3.8 nM for the D- and 0.89 nM for the S-site. The densities (B_max values) of both binding sites were 1.7 pmol/mg protein for the D-site and 0.79 pmol/mg protein for the S-site. [³H]LSD binding to the D-site was reversible and reached equilibrium in about 30 min. Pharmacological displacement studies display a high binding affinity of the putative natural agonist dopamine to the D-site (K_i = 22 nM). The most potent displacers of D-site binding were lisuride, (+)-bromocriptine, chlorpromazine, S(+)-butaclamol, and 6,7-ADTN. The [³H]LSD labelled D-site seems to be G-protein coupled, since addition of the stable GTP analogue GTPγS or NaCl to the incubation medium evoked a decrease of specific [³H]LSD binding to the D-site.     

Modulation of [3H]-glutamate binding by serotonin in the rat hippocampus: an autoradiographic study.

Serotonin (5-HT) added in vitro (10 microM) increased [3H]-glutamate specific binding in the rat hippocampus, reaching statistical significance in layers rich in N-Methyl-D-Aspartate sensitive glutamate receptors. This effect was explained by a significant increase in the apparent affinity of [3H]-glutamate when 5-HT is added in vitro. Two days after lesion of serotonergic afferents to the hippocampus with 5,7-Dihydroxytryptamine [3H]-glutamate binding was significantly decreased in the CA3 region and stratum lacunosum moleculare of the hippocampus, this reduction being reversed by in vitro addition of 10 microM 5-HT. The decrease observed is due to a significant reduction of quisqualate-insensitive (radiatum CA3) and kainate receptors (strata oriens, radiatum, pyramidal of CA3). Five days after lesion [3H]-glutamate binding increased significantly in the CA3 region of the hippocampus but was not different from sham animals in the other hippocampal layers. Two weeks after lesion [3H]-glutamate binding to quisqualate-insensitive receptors was increased in all the hippocampal layers, while kainate and quisqualate-sensitive receptors were not affected. These data are consistent with the possibility that 5-HT is a direct positive modulator of glutamate receptor subtypes.     

In vitro metabolism of [3H]-peptide leukotrienes in human and ferret lung: a comparison with the guinea pig

Fragmented lung tissue prepared from human, ferret and guinea pig converted [3H]-leukotriene C4 ([3H]-LTC4) to [3H]-LTD4 and [3H]-LTE4. [3H]-LTD4 was the major product recovered from incubations with human and guinea pig lung, whereas [3H]-LTE4 was the predominant metabolite in ferret. Kinetic analysis in human lung yielded a much faster rate for the conversion of [3H]-LTC4 to [3H]-LTD4+E4 than for the conversion of [3H]-LTD4 to [3H]-LTE4. In all three species serine-borate complex blocked the metabolism of [3H]-LTC4, whereas L-cysteine blocked the metabolism of [3H]-LTD4. These studies demonstrate that guinea-pig and human lungs have a similar metabolic pattern and capacity which is dissimilar to the ferret.     

[3H]2-nitroimipramine: A selective “slowly-dissociating” probe of the imipramine binding site (“serotonin transporter”) in platelets and brain

Previous studies have demonstrated a close functional and structural relationship between the “high affinity” binding site for [³H]imipramine and the presynaptic and platelet uptake site(s) for serotonin. Recently we have synthesized several nitro derivatives of imipramine which have a very high affinity for the imipramine binding site and which dissociate very slowly when incubations are performed at 0–4°C. In this report, we describe the characteristics of [³H]2-nitroimipramine binding to platelet and brain membranes. Our results support the relative utility of this ligand for studying the impramine binding site (serotonin transporter) since this analogue has both a higher affinity and specific activity than [³H]imipramine. [³H]2-Nitroimipramine by virtue of its extremely slow dissociation rate should be a valuable tool in subsequent characterization and purification of the serotonin uptake or transport site.     

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of [3H]dextromethorphan and sigma ligands to guinea pig brain.

The DM1/sigma 1 site binds dextromethorphan (DM) and sigma receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of [3H]dextromethorphan, [3H]3-(-3-Hydroxyphenyl)-N-(1-propyl)piperidine and (+)-[3H]1,3-Di-o-tolyl-guanidine ([3H]DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM Ki values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM1/sigma 1 site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed Ki values of 9-13 and 3-4 microM respectively against the three labeled ligands. These results, the broad specificity of the DM1/sigma 1 binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor. These findings may have important implications for the understanding of the therapeutic, side effects and toxicity of several neurotropic drugs.     

Identification of leukotriene D4 receptor binding sites in guinea pig lung homogenates using [3H]leukotriene D4

We have characterized [3H]leukotriene D4 binding to guinea pig lung homogenates. Both biphasic dissociation kinetics and curvilinear Scatchard plots indicated the presence of [3H]leukotriene high and low affinity states of the binding sites. The rank order of potency for the competition study was leukotriene C4 = leukotriene D4 greater than leukotriene E4 much greater than arachidonic acid, and for their contractile effect on lung strips was leukotriene C4 = leukotriene D4 = leukotriene E4 much greater than arachidonic acid. FPL-55712 was the only other agent tested that inhibited binding. These results suggest that binding of [3H]leukotriene D4 to the homogenate is consistent with its binding to specific leukotriene D4 receptor sites.     

Characterization of [3H]-forskolin binding sites in young and adult rat brain cortex: identification of suramin as a competitive inhibitor of [3H]-forskolin binding

Little is know about forskolin binding in the rat brain during ontogenetic development. For this paper, we have characterized specific binding sites for [3H]-forskolin in cerebrocortical membranes from young (12-day-old) and adult (90-day-old) rats. High-affinity, as well as super-high-affinity, [3H]-forskolin binding sites were detected in samples from both age groups tested, and the binding parameters of these sites differed significantly. Whereas the number of high-affinity [3H]-forskolin binding sites was higher by about 50% in adult than in young rats, their affinity was markedly (about 4 times) lower. In the presence of AlF4-, the number high-affinity [3H]-forskolin binding sites in samples from young rats rose to the level determined in samples from adult animals, and the number of super-high-affinity sites considerably increased in both age groups. The different characteristics of [3H]-forskolin binding found in cerebrocortical membranes from young and adult rats may be closely related to markedly diminished adenyl cyclase activity in preparations from adult animals. Results of our experiments with suramin indicated that this drug may act as a competitive inhibitor of [3H]-forskolin binding.     

Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: [3H]chlorpromazine labels homologous residues in the beta and delta chains

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled beta chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by [3H]chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the beta chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain [Giraudat, J., Dennis, M., Heidmann, T., Chang, J. Y., & Changeux, J.-P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2719-2723]. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.