Sites In Brain Membranes Research Articles

The hypotensive effect of BMY 7378 involves central 5-HT 1A receptor stimulation in the adult but not in the young rat

Stimulation of central 5-hydroxytryptamine-1A (5-HT(1A)) receptors produces hypotension and bradycardia. We describe BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9 dione) effects in cardiovascular function and [(3)H] 8-OH-DPAT (8-hydroxy-2-(di-n-propyl-amino) tetralin) binding sites in rat brain of different ages. BMY 7378 was administered to anesthetized male Wistar rats (1, 3 and 6 months old) and blood pressure and heart rate were continuously recorded. Saturation of [(3)H] 8-OH-DPAT binding to 5-HT(1A) sites in brain membranes was determined. Basal diastolic blood pressure increased with age, 85 +/- 2, 106 +/- 3, and 113 +/- 2 mmHg for 1-, 3- and 6-month-old rats, respectively (p <0.05 among groups). BMY 7378 induced significant dose- and age-dependent hypotension. The selective 5-HT(1A) receptor antagonist, WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]N-(2-pyridinyl) cyclohexanecarboxamide), antagonized BMY 7378 effects in 6 month-old but not in younger rats. [(3)H] 8-OH-DPAT binding sites decreased in hippocampi and brainstem with maturation. Data suggest that BMY 7378 is a hypotensive agent in the rat, but that its actions are mediated, in part, by central 5-HT(1A) receptor stimulation in the adult and by a nonserotonergic mechanism in the young rat.

Binding crevice for TT-232 in a homology model of type 1 somatostatin receptor

Somatostatin receptor type 1 was modelled based on the atomic structure of bovine rhodopsin. Possible ways of binding interaction between somatostatin receptor type 1 and TT-232, a cycloheptapeptide analogue of somatostatin with broad therapeutic potential, were analysed by molecular docking. The twelve TT-232 conformations, obtained by NMR measurements in H 2O–D 2O mixture, were similar, disclosing a consensus backbone conformation. Several residues interacting with TT-232, such as Val133, Asp137 (helix 3), Arg197 (helix 4), Phe287, Gln291, Asn294 (helix 6), Ser305, and Tyr313 (helix 7), were found. In accordance, in vitro binding experiments indicated high-affinity binding of TT-232 to 125I labelled somatostatin sites in brain membranes. The single binding crevice obtained by docking may allow the design and discovery of new peptidomimetics of TT-232 in the future.

Studies of the biogenic amine transporters. 10. Characterization of a novel cocaine binding site in brain membranes prepared from dopamine transporter knockout mice.

Previous work suggested that the cocaine analog [(125)I]RTI-55 labels a novel binding site in rat brain membranes, which is not associated with the dopamine (DA), serotonin (5-HT), or norepinephrine (NE) transporters [Rothman et al. 1995 J Pharmacol Exp Ther 274:385-395]. Here, we tested whether this site is a product of the DA transporter (DAT) gene. We used a T-antigen knock-in at the DAT gene that results in an effective DAT knock-out (KO) confirmed by Southern blot, DAT immunohistochemistry, and [(125)I]RTI-55 ligand binding. Brain membranes were prepared from frozen whole brain minus caudate of wild-type (WT) B6/Sv129, +/+ and minus sign/minus sign (KO) mice. KO mice were used at approximately 23 days of age. Binding surface analysis of [(125)I]RTI-55 binding to membranes prepared from the brains of WT mice, with 100 nM citalopram to block binding to the 5-HT transporter (SERT), revealed two binding sites: the DAT and a second site, replicating previous studies conducted with rat brains. In the absence of the DAT (minus sign/minus sign mice), binding surface analysis demonstrated that [(125)I]RTI-55 labeled two sites: the NET and a second site called site "X." Structure-activity studies of site "X" demonstrated that high-affinity ligands for the DAT, NET, and SERT have low or negligible affinity for site "X." The relatively high density of site "X" in brain membranes and the fact that the K(i) values of cocaine and cocaethylene for site "X" are in the range achieved in the brain following cocaine administration suggests that site "X" could contribute to the pharmacological or toxicological effects of cocaine. Further progress in delineating the function of site "X" will depend on developing potent and selective agents for this site.

Widespread Distribution of Binding Sites for the Novel Ca2+-mobilizing Messenger, Nicotinic Acid Adenine Dinucleotide Phosphate, in the Brain

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing agent in invertebrate eggs that has recently been shown to be active in certain mammalian and plant systems. Little, however, is known concerning the properties of putative NAADP receptors. Here, for the first time, we report binding sites for NAADP in brain. In contrast to sea urchin egg homogenates, [(32)P]NAADP bound reversibly to multiple sites in brain membranes. The rank order of potency of NAADP, 2',3'-cyclic NAADP and 3'-NAADP in displacing [(32)P]NAADP was, however, the same in the two systems and in agreement with their ability to mobilize Ca(2+) from homogenates. These data indicate that [(32)P]NAADP likely binds to receptors mediating Ca(2+) mobilization. Autoradiography revealed striking heterogeneity in the distribution of [(32)P]NAADP binding sites throughout the brain. Our data strongly support a role for NAADP-induced Ca(2+) signaling in the brain.

Diazepam-potentiated [ 3H]phenytoin binding is associated with peripheral-type benzodiazepine receptors and not with voltage-dependent sodium channels

In the presence of diazepam, [ 3H]phenytoin binds with high affinity to brain membranes. The present experiments examined whether this high affinity [ 3H]phenytoin-binding site co-localized with the standard [ 3H]phenytoin-binding site on the voltage-dependent sodium channel (VDSC). Veratridine, a pharmacological activator of the voltage-dependent sodium channel, that inhibits standard [ 3H]phenytoin binding, failed to affect the high affinity diazepam-potentiated [ 3H]phenytoin binding in brain membranes, suggesting that the potentiated binding interaction resides at a site distinct from the voltage-dependent sodium channel. This possibility was confirmed by anion exchange chromatography of digitonin-solubilized rat brain membranes, as diazepam-potentiated high affinity [ 3H]phenytoin binding eluted in column fractions that were distinct from [ 3H]saxitoxin-defined voltage-dependent sodium channels. To examine whether diazepam-potentiated [ 3H]phenytoin binding might be associated with other ‘classic’ benzodiazepine receptor sites, we tested whether specific ligands for benzodiazepine receptors would either produce or block potentiated [ 3H]phenytoin binding. Neither agonists, nor antagonists, of the high affinity central-type benzodiazepine receptor affected potentiated [ 3H]phenytoin binding, suggesting that the high affinity potentiated binding site is not likely associated with central benzodiazepine receptors. Peripheral-type benzodiazepine receptor agonists, however, did potentiate [ 3H]phenytoin binding, and a specific receptor antagonist (PK11195) attenuated the potentiation seen with diazepam. Overall, these data illustrate that [ 3H]phenytoin interacts with a novel site in brain membranes that is distinct from the voltage-dependent sodium channel and is allosterically revealed by peripheral-type, but not central-type, benzodiazepine receptor agonists.

Distinct Specificity for Corticosteroid Binding Sites in Amphibian Cytosol, Neuronal Membranes, and Plasma

To address mechanisms of corticosteroid action, one needs tools for distinguishing between the major classes of corticosteroid binding sites: neuronal membrane-associated receptors, intracellular ligand-activated transcription factors, and corticosteroid binding globulins (CBG) in plasma. We characterized the binding parameters for three classes of binding sites in an amphibian, Ambystoma tigrinum, and found that each class had a distinct pharmacological specificity. Equilibrium saturation and kinetic experiments indicated that [3H]corticosterone binds to neuronal membranes with high affinity (Kd ≈ 0.37 nM). Aldosterone and two synthetic ligands for mammalian intracellular receptors, dexamethasone and RU486, displayed low affinity for brain membrane sites. In cytosol prepared from brain and liver, [3H]corticosterone bound to a single class of receptors with high affinity (Kd ≈ 0.75 and 4.69 nM, respectively) and the rank order potencies for steroid inhibition of [3H]corticosterone binding was RU486 > dexamethasone ≈ corticosterone > aldosterone. In kidney and skin cytosol, [3H]corticosterone binding was best fit with a model having a high-affinity and a lower-affinity site; these sites are not consistent with the pharmacology of mammalian Type I (MR) and Type II (GR) receptors. [3H]Corticosterone also bound to presumed CBG in plasma with high affinity (Kd ≈ 2.7 nM), but dexamethasone and androgens bound to plasma CBG with equivalently high affinity. These data demonstrate that pharmacological specificity can be a useful tool for distinguishing corticosteroid binding to different classes of binding sites. These data also indicate that there may be marked species differences in the specificity of corticosteroid binding sites.

P-207 - High-affinity hemichoIinium-3 binding sites in brain membranes of rat neonates: upregulation by nicotine treatment

P-207 - High-affinity hemichoIinium-3 binding sites in brain membranes of rat neonates: upregulation by nicotine treatment

Arachidonylethanolamide (anandamide) binds with low affinity to dihydropyridine binding sites in brain membranes

The purpose of this study was to explore the hypothesis that the dihydropyridine (DHP) binding site of the L-type calcium channel is a high affinity binding site for the cannabimimetic arachidonylethanolamide (AEA). Binding affinities were determined from competition isotherms using the DHP analog [ 3H]PN-200. AEA competed for [ 3H]PN-200 binding with a K 1 of 40 ± 4 μM. Inclusion of phenylmethylsulfonyl fluoride to inhibit an amidohydrolase that converts AEA to arachidonic acid had little effect on the K 1 of AEA (48 ± 6 μM). Arachidonic acid had a slightly higher K 1 (120 ± 11 μM) and other N-acylethanolamides examined (linolenylethanolamide, dihomo-γ-linolenylethanolamide, docosatetraenylethanolamide, and palmitoylethanolamide) had no effect on [ 3H]PN-200 binding at concentrations as high as 10 μM. Our conclusions are that AEA binds to the DHP binding site with relatively low affinity and its conversion to arachidonic acid is not required for binding.

Characterization of the μ and δ Opioid Receptors in the Brain of the C57BL/6 and DBA/2 Mice, Selected for Their Differences in Voluntary Ethanol Consumption

Genetically determined differences in the activity of the hypothalamic beta-endorphin system have been demonstrated between the C57BL/6 (alcohol-preferring) and DBA/2 (alcohol-aversive) inbred strains of mice. The present studies examined the distribution and density of the mu and delta receptors in specific brain regions that may mediate the rewarding and reinforcing effects of ethanol, using quantitative autoradiography and the specific mu agonist FK 33-824 and delta agonist DPDPE, in their iodinated form. 125I-FK 33-824 recognizes a high affinity binding site in brain membrane preparations from both the C57BL/6 (Kd = 1.37 +/- 0.22 nM; Bmax = 80 +/- 12.3 fmol/mg protein) and DBA/2 mice (Kd = 1.02 +/- 0.16 nM; Bmax = 39.5 +/- 9.6 fmol/mg protein), whereas 125I-DPDPE binds to a high affinity binding site in brain membranes from both the C57BL/6 (Kd = 1.08 +/- 0.34 nM; Bmax = 24.4 +/- 4.5 fmol/mg protein) and DBA/2 mice (Kd = 0.68 +/- 0.24 nM; Bmax = 15.3 +/- 3.7 fmol/mg protein). The autoradiographic studies demonstrated differences in the density of the mu opioid receptors between the two strains of mice in brain nuclei that are not directly related to the brain reward system. However, strain-related differences in the density of delta opioid receptors were observed in regions of the limbic system known to mediate the positive reinforcing effects of many drugs of abuse. The density of delta receptors was significantly higher in the ventral tegmental area and nucleus accumbens of the C57BL/6 mice. The results of the present study support the hypothesis that genetically determined differences exist in the density of opioid receptors in distinct regions of the brain between the C57BL/6 and DBA/2 inbred strains of mice, which may play a role in controlling their voluntary ethanol consumption.

Characterization of adenosine receptors in Mullus surmuletus

The intent of the present study was to investigate adenosine receptor sites in brain membranes of the saltwater teleost fish, Mullus surmuletus, using the A 1 receptor selective agonist, [ 3H]CHA, and A 2a receptor selective agonist [ 3H]CGS 21680. The A 1 selective agonist, [ 3H]CHA, bound saturably, reversibly and with high affinity to a single-class of binding sites (K d 1.47 nM; B max 100–190 fmol/mg protein, dependent on fish length). The A 2a selective agonist, [ 3H]CGS 21680, also bound saturably, reversibly and with relative high affinity to a single-class of binding sites (K d 44.2 nM; B max 150–300 fmol/mg protein dependent on fish length). In equilibrium competition experiments, adenosine analogous, NECA, CGS 21680, CHA, CPA, S-PIA, R-PIA, CPCA, DPMA, and xanthine antagonists, DPCPX, XAC, and THEO all displaced [ 3H]CHA and [ 3H]CGS 21680 specifically bound to brain membranes from Mullus surmuletus. Specific binding of both [ 3H]CHA and [ 3H]CGS 21680 was inhibited by GDPβS. For [ 3H]CHA the IC 50 value was 2.5 ± 0.1 μM, while for [ 3H]CGS 21680 the IC 50 value was 7.7 ± 0.3 μM. Our results indicate that the high affinity binding sites for [ 3H]CHA have some pharmacological characteristics of mammalian A 1 adenosine receptors, while the binding sites for [ 3H]CGS 21680 appear to be virtually identical to the binding sites for [ 3H]CHA.

3H]Aniracetam Binds to Specific Recognition Sites in Brain Membranes

[3H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na+ independent, was still largely detectable at low temperature (4 degrees C), and underwent rapid dissociation. Scatchard analysis of [3H]aniracetam binding revealed a single population of sites with an apparent KD value of approximately 70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [3H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [3H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [3H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37 degrees C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [3H]aniracetam binding in unwashed membranes. High levels of [3H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective block of recombinant glur6 receptors by NS-102, a novel non-NMDA receptor antagonist

The diversity of neuronal glutamate receptors continues to increase with the discovery of multiple subunits and subunit families. The significance of this potential receptor heterogeneity is unknown because pharmacological tools that could clearly distinguish between different structural isoforms have not yet been identified. A novel glutamate receptor antagonist, 5-nitro-6,7,8,9-tetrahydrobenzo [ g]indole-2,3-dione-3-oxime (NS-102), has been shown previously to selectively block the low affinity [ 3H]kainate binding site in rat brain. We have examined the effect of NS-102 on receptors expressed in fibroblasts from either glur6 subunits or a combination of glurB and glurD (glurB/D receptors). NS-102 (3 μM) reduced currents mediated by glur6 receptors and had very little effect on currents mediated by glurB/D receptors. The binding of [ 3H]kainate to glur6 receptors showed properties similar to those of the brain low affinity [ 3H]kainate binding site, and NS-102 inhibited specific binding to glur6 receptors with a potency nearly identical to those sites in brain membranes. Our findings suggest that NS-102 will be useful in identifying the functional role of native receptors containing a glur6 subunit.

Binding characteristics of the novel highly selective delta agonist, [ 3H]Ile 5,6deltorphin II

Following the description of the [ 3H]deltorphin II, it has been reported that the modification of deltorphin II with the substitution of Val 5,6 residues by the more hydrophobic lle 5,6 residues leads to an increased affinity and selectivity. The lle 5,6deltorphin II (Tyr-D-Ala-Phe-Gly-lle-lle-Gly-NH 2) was tritiated by catalytic dehalogenation and labelled rat brain membrane sites with a K d value of 0.40 nM and a B max of 121 fmol/mg protein. Competition binding experiments with various unlabelled subtype specific opioid receptor ligands resulted in μ δ and κ δ selectivity ratios of 2400 and 18 000 respectively. Due to its high δ receptor affinity, δ selectivity and very low non-specific binding (<20%), [ 3H]lle 5,6deltorphin II, is a very useful tool for the identification and characterisation of δ opioid receptors.

Identification of two regions of beta G spectrin that bind to distinct sites in brain membranes.

This study analyzed the complex interactions of intact spectrin with bovine brain membranes by evaluating membrane associations of defined regions of beta G spectrin, the subunit responsible for high affinity membrane binding. Two regions of beta G spectrin were expressed in bacteria and demonstrated to contain fully functional binding site(s) for a subset of spectrin-binding sites in brain membranes depleted of peripheral proteins. One region, located near the NH2 terminus, was comprised of 106-residue repeats and required repeats 2-7 for full activity. The other binding domain was located at the COOH terminus, which is the most variable between beta G and beta R spectrins, is distinct from the 106-residue repeats, and contains a pleckstrin homology domain. NH2-terminal beta spectrin polypeptides interacted with a membrane site(s) that recognized both brain and erythrocyte isoforms of spectrin, was inhibited by calcium/calmodulin, and was not blocked by the COOH-terminal polypeptide. The COOH-terminal region associated with a membrane site(s) that was specific for brain spectrin, was not inhibited by calcium/calmodulin, and was not blocked by the NH2-terminal polypeptide. These observations demonstrate membrane association of spectrin with at least two independent sites, which differ with regard to regulation by calcium/calmodulin and in selectivity for spectrin isoforms.

Photoactivatable 2-(4'-azidotetrafluorophenyl)-5-tert-butyl-1,3-dithiane-bis- sulfone and related compounds as candidate irreversible probes for the GABA-gated chloride channels.

Syntheses of 2-substituted photoactivatable derivatives of 5-tert-butyl-1,3-dithiane and their oxidized bis-sulfone derivatives are described with the aim of developing original photoaffinity probes for the GABA-gated chloride channel. Diazocyclohexadienone as well as fluorinated arylazido derivatives were chosen as photosensitive moieties. The dithiane molecules were synthesized by condensation of 2-tert-butylpropane-1,3-dithiol with the appropriate substituted benzaldehydes. The diazocyclohexadienonyl derivatives were synthesized either by diazotization of the corresponding dithiane arylamine precursors (compounds 1, 2, 17, and 18) or by diazotization of the bis-sulfone dithiane arylamine (compounds 19 and 20). The reversible binding properties of the photosensitive probes were established on bovine cortex P2 membranes by displacement of [3H]-tert-butylbicycloorthobenzoate. While the bromo-substituted diazocyclohexadienoyl dithiane derivatives (compounds 1 and 2) exhibited a Ki of about 2-4 microM 2-(4'-azidotetrafluorophenyl)-5-tert-butyl-1,3-dithiane-bis-sulfon e (compound 3) gave a Ki of 0.2 microM. On irradiation, probe 3 produced a 25% irreversible loss of TBOB binding sites in brain membranes. Moreover, this loss was fully protectable by TBOB, demonstrating the specificity of the photochemical inactivation by compound 3 for the convulsant site of the GABAA receptor.

Haloperidol treatment differentially regulates [ 3H]DTG and [ 3H](+)-3-PPP labeled σ binding sites

The effect of repeated haloperidol administration on σ binding sites in brain membranes was assessed using [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP) and [ 3H]1,3-di-o-tolylguanidine (DTG). Administration of haloperidol (1 mg/kg, i.p.) to guinea pigs for 14 consecutive days followed by a 4 day drug-free period prior to sacrifice resulted in 75% and 6% decreases in the specific binding of [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and [ 3H]1,3-di-o-tolylguanidine, respectively, when measured using a single concentration (2 nM) of radioligand. Scatchard analysis revealed a reduction in both the maximum number of [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding sites and the affinity of these sites for the radioligand; the potency of 1,3-di-o-tolylguanidine to inhibit [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding was also reduced. In parallel studies, the potency of 1,3-di-o-tolylguanidine to inhibit [ 3H]1,3-di-o-tolylguanidine binding was unaffected by haloperidol treatment, but the potency of (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine against [ 3H]1,3-di-o-tolylguanidine was reduced 3-fold. Phenytoin, which increased (10-fold) the potency of dextromethorphan to inhibit [ 3H]1,3-di-o-tolylguanidine binding in control membranes, had no effect in membranes obtained from haloperidol-treated animals. The reduction in [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding was dependent upon the duration of the drug-free period and amounted to 73% and 25% in brain membranes prepared from animals sacrificed 14 days and 28 days, respectively, following cessation of drug treatment. Repeated administration of other antipsychotic and σ agents including DTG, dextromethorphan, spiperone, chlorpromazine and clozapine had no effect on [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine or [ 3H]1,3-di-o-tolylguanidine binding. These findings suggest that repeated haloperidol administration selectively regulates [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding in guinea pig brain membranes. However, the observations that repeated drug treatment resulted in (1) reduced affinity of (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine for [ 3H]1,3-di-o-tolylguanidine binding sites, and (2) a loss of the ability of phenytoin to increase dextromethorphan's potency against [ 3H]1,3-di-o-tolylguanidine, suggest that [ 3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and [ 3H]1,3-di-o-tolylguanidine binding sites are distinct but functionally coupled.

Opioid binding sites in jerboa ( Jaculus orientalis) brain: a biochemical comparative study in the awake-active and induced hibernating states

1. 1. Using tritiated ligands [ 3H]DADLE, [ 3H]DAGO, [ 3H]EKC and [ 3H]Bremazocine, we have demonstrated the presence of δ, μ and κ sites in brain membranes from jerboa ( Jaculus orientalis), a desert rodent and a true hibernator. 2. 2. A comparative study was realized in the case of the induced hibernating state, indicating a reduction of binding capacities during the hibernation state. 3. 3. Using radioimmunoassay, the endogenous pentapeptide methionine-enkephalin (Met-enk) was evaluated in different areas of jerboa brain in comparison with the effect of induced hibernation on the level of Met-enk. 4. 4. A thermodynamic analysis of the effect of temperature on the binding of opioids, indicates that the hibernating and the active state are energetically different. ΔG°, ΔH° and ΔS° were calculated. A break of the Van't Hoff plot was observed in the active state at 15°C, indicating a possible transition state of membranous phospholipids and/or proteins. 5. 5. The role of phospholipids was studied, using the effect of phospholipase A 2 and membrane reconstitution. Phospholipids play a key role in the opioid binding.

Characterization of binding sites for [ 3H]-DTG, a selective sigma receptor ligand, in the sheep pineal gland

Specific binding sites for [ 3H]-1,3 di-ortho-tolylguanidine ([ 3H]-DTG), a selective radiolabeled sigma receptor ligand, were detected and characterized in sheep pineal gland membranes. The binding of [ 3H]-DTG to sheep pineal membranes was rapid and reversible with a rate constant for association (K +1) at 25°C of 0.0052 nM −1 .min −1 and rate constant for dissociation (K −1) 0.0515 min −1, giving a K d ( K −1 K +1 ) of 9.9 nM. Saturation studies demonstrated that [ 3H]-DTG binds to a single class of sites with an affinity constant (K d) of 27 ± 3.4 nM, and a total binding capacity (B max) of 1.39 ± 0.03 pmol/mg protein. Competition experiments showed that the relative order of potency of compounds for inhibition of [ 3H]-DTG binding to sheep pineal membranes was as follows: trifluoperazine = DTG> haloperidol> pentazocine> (+)-3-ppp > (+/−) SKF 10,047. Some steroids (testosterone, progesterone, deoxycorticosterone) previously reported to bind to the sigma site in brain membranes were very weak inhibitors of [ 3H]-DTG binding in the present study. The results indicate that [ 3H]-DTG binding sites having the characteristics of sigma receptors are present in sheep pineal gland. The physiological importance of these sites in regulating the synthesis of the pineal hormone melatonin awaits further study.

Nicotinic Modulation of [3H]Dopamine Release from Striatal Synaptosomes: Pharmacological Characterisation

Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. We have compared the effects of a number of nicotinic agonists and antagonists on a perfused synaptosome preparation preloaded with [3H]dopamine. (-)-Nicotine, acetylcholine, and the nicotinic agonists cytisine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), at micromolar concentrations, stimulated the release of [3H]dopamine from striatal nerve terminals. Carbamylcholine was a much weaker agonist. The actions of (-)-nicotine, cytisine, and DMPP were inhibited by low concentrations of the nicotinic antagonists dihydro-beta-erythroidine, mecamylamine, pempidine, and neosurugatoxin; alpha-bungarotoxin was without effect, and extending the time of exposure to this toxin resulted in only very modest inhibition. This pharmacology points to a specific nicotinic receptor mechanism that is clearly distinct from that at the neuromuscular junction. Atropine failed to antagonise the effects of acetylcholine and carbamylcholine, suggesting that no muscarinic component is involved. The nicotinic receptor ligands (-)-[3H]nicotine and 125I-alpha-bungarotoxin bound to specific sites enriched in the synaptosome preparation. Drugs tested on the perfused synaptosomes were examined for their ability to interact with these two ligand binding sites in brain membranes. The differential sensitivity to the neurotoxins alpha-bungarotoxin and neosurugatoxin of the 125I-alpha-bungarotoxin and (-)-[3H]nicotine binding sites, respectively, leads to a tentative correlation of the (-)-[3H]nicotine site with the presynaptic nicotinic receptor on striatal nerve terminals.

Distribution of basic fibroblast growth factor binding sites in various tissue membrane preparations from adult guinea pig

In order to localize a rich source of basic FGF receptor, we examined the distribution of basic FGF binding sites in brain, stomach, lung, spleen, kidney, liver and intestine membrane preparations from adult guinea pig. Comparative binding studies using iodinated basic FGF showed that a specific binding was detected in all the membrane preparations tested. Scatchard plots from iodinated basic FGF competition experiment with native basic FGF in various membrane preparations, suggested the presence of one class of binding sites in some tissues such as liver, kidney, spleen, lung, stomach, and intestine with an apparent dissociation constant (appKD) value ranging from 4 to 7.5 nM and the existence of a second class of higher affinity sites in brain membranes with appKD value of 15 pM. Characterization of these basic FGF high affinity interaction sites was performed using a cross-linking reagent. These results show for the first time that specific interaction sites for basic FGF are widely distributed, suggesting that this growth factor might play a role in the physiological functions of a number of adult organs.