Sites In Brain Membranes Research Articles

Characteristics of sodium-induced increase in opiate antagonist binding sites in rat brain membranes

Sodium decreases agonist binding and increases antagonist binding to opiate receptor sites in brain membranes. This study characterizes in detail the 20–40% increase in [ 3H]naloxone binding caused by sodium. This increase in binding was specific to sodium (not mimicked by potassium or lithium) and was maximal at 10 mM NaCl. The sodium effect was reversible: washing membranes free of sodium restored binding to normal. Sodium increased B max, not K D, of [ 3H]naloxone binding. The sodium-induced increased binding was not inhibited by either N-ethylmaleimide (NEM) or phospholipase A 2 at concentrations which inhibit 50–85% of normal [ 3H]naloxone binding. In NEM-treated membranes, the effect of sodium on increasing naloxone binding was actually increased. The regional distribution of these sodium-dependent sites were different from normal naloxone sites. These results suggest that physiological concentrations of sodium expose naloxone sites which differ in biochemical properties from naloxone sites assayed in the absence of sodium.

Structural and pharmacological differences between codfish and rat brain alpha 1-adrenergic receptors revealed by photoaffinity labeling with 125I-APDQ.

The photoaffinity probe 125I-APDQ has been used to characterize alpha 1-receptor peptides in the cod and rat brains. In the cod brain a major specific peptide of Mr = 68,000 could be covalently labeled by 125I-APDQ as revealed by SDS-PAGE. In the rat brain a specific peptide with Mr = 77,000 was instead labeled. When a number of adrenergic agonists and antagonists were tested for their ability to protect the labeling by 125I-APDQ their potencies were those expected for alpha 1-receptors in both species. The ligand binding peptide in the cod brain also distinguished between stereoisomers of epinephrine as expected for a physiological receptor. However, there was a distinct difference between the cod and rat alpha 1-receptor in that the beta-agonist 1-isoprenaline was equipotent to 1-norepinephrine in the cod whereas it was less potent in the rat. The protecting ability of the tested agents were also matched by their ability to displace the alpha 1-adrenergic ligand 3H-prazosin from alpha 1-receptor binding sites in brain membranes from both species. Thus, the codfish alpha 1-receptor seems to be different from mammalian alpha 1-receptors both structurally and pharmacologically.

Characterization of the 5-HT IB recognition site in rat brain: Binding studies with (−)[ 125I]Iodocyanopindolol

(−)[ 125I]Iodocyanopindol ([ 125I]CYP) labels rat brain membrane sites which display high affinity for several serotonergic and β-adrenergic compounds. The binding of [ 125I]CYP to these serotonergic recognition sites was evaluated in the presence of 30 μM (−)isoprenaline in order to suppress binding to β-adrenoceptors. [ 125I]CYP binds in rat cortex membranes rapidly, reversibly and stereoselectivity to a finite number of recognition sites: B max = 180 fmol/mg, K D = 230 pM. Similar affinity values of [ 125I]CYP were obtained in membranes from rat hippocampus and striatum. Kinetic, saturation and competition experiments suggest that under these conditions [ 125I]CYP binds to a single serotonergic recognition site named 5-HT 1B. The pharmacological profile of 5-HT 1B sites is characteristic of a 5-HT 1 binding site and shows the following rank order of affinity for agonists: RU 24969, (5-methoxy-3-[1,2,3,6-tetra-hydropyridin-4-yl]1H-indole) > 5-CT, (5-carboxamidotryptamine) > 5-HT, (5-hydroxytryptamine, serotonin) > 5-OCH 3-T, (5-methoxytryptamine) ⪢ 2-CH 3-5-HT, (2-methylserotonin) > 8-OH-DPAT, (8-hydroxy-2-(di-n-propylamino)-tetralin). The rank order of affinity for antagonists is: (±)ICYP, ((±)-3-I-cyano-pindolol) > (−)21-009, (4-[3-ter-butyl-amino-2-hydroxy-propoxy]-indol-2-carbonic acid isopropyl ester) > (+)21-009 > (−)propranolol > metitepin > (−)pindolol ⪢ ketanserin > spiroperidol > mesulergine. 5-HT 1B recognition sites display low affinity for selective β 1- and β 2-adrenoceptor antagonists, e.g. atenolol, betaxolol, ICI 89-406 and ICI 118-551. The low affinity of 5-HT 1B recognition sites for some 5-HT 1A, 5-HT 1C and 5-HT 2 selective compounds (e.g. 8-OH-DPAT, mesulergine, ketanserin) suggests that 5-HT 1B recognition sites are pharmacologically different from 5-HT 1A, 5-HT 1C and 5-HT 2 recognition sites.

Regulation of calcium antagonist binding sites in brain membranes: Effect of peptides

Regulation of calcium antagonist binding sites in brain membranes: Effect of peptides

3H]Oxotremorine-M binding to membranes prepared from rat brain and heart: Evidence for subtypes of muscarinic receptors

[ 3H]Oxotremorine-M has been used as a ligand to label muscarinic binding sites on membranes prepared from rat fore-brain and heart. In rat brain membranes, two binding sites could be identified: a high affinity low capacity site and a low affinity high capacity site. In contrast, only a high affinity site could be labelled in heart membranes. The potency order of agonists for the high affinity site in brain membranes and heart membranes was the same, suggesting that these binding sites represent the same subtype of muscarinic receptors. However, the affinity of pirenzepine for the brain high affinity site was higher than that for the heart high affinity site suggesting that these sites may represent different receptor populations. The potency order of agonists for the high affinity and low affinity sites in brain membranes were significantly different suggesting that these binding sites represent pharmacologically distinct binding sites. GTP abolished the high affinity sites in heart and brain membranes, but the low affinity site in brain membranes appeared unaffected. These results are consistent with the hypothesis proposing subtypes of muscarinic receptors.

Interaction of dextrorotatory opioids with phencyclidine recognition sites in rat brain membranes

The potencies of several dextrorotatory opioids, including four pairs of enantiomers, as inhibitors of specific [ 3H]PCP binding to rat brain synaptic membranes has been determined. Of the compounds tested unlabeled phencyclidine (PCP) was the most potent followed by (−)− cyclazocine > dextrorphan > (+) ketamine > (+) cyclazocine > (+)− SKF10,047 > levorphanol > dextromethorphan > (−) SKF10,047 > (−)− ketamine > (±) pentazocine and > (±) ethylketocyclazocine. The opiate mu receptor ligands, morphine, naloxone and naltrexone were virtually inactive as competitors of specific [ 3H]PCP binding. Unlike the stereostructural requirements for opiate mu receptors where activity resides predominantly in the levorotatory enantiomers, the present results support the contention that binding to the [ 3H]PCP labeled recognition site may reside in either the levorotatory or the dextrorotatory enantiomer. The specific binding of [ 3H]PCP which was defined as total binding minus that occurring in the presence of 10μM dextrorphan was found to be of a high affinity, saturable, reversible and sensitive to thermal degradation. These results suggest that certain dextrorotatory morphian derivatives may prove to be useful probes in further investigations of the molecular characteristics of the [ 3H]PCP binding site in brain membrane preparations.

Theoretical and experimental studies of drug-receptor interactions: Determinants for recognition of 5-hydroxytryptamine analogs

Experimental studies on 5-hydroxytryptamine (5-HT) and its congeners have shown these compounds to interact with the same receptors in peripheral tissues and in brain. To evaluate the importance of the relative position of the 5-HT-like recognition elements at these receptors, we studied two compounds structurally related to 5-HT in which the structural elements involved in receptor recognition are positioned differently from 5-HT: 5-hydroxyaminotetrahydrobenzindole (FHATHBIN) in which the position of the side chain is fixed with respect to the indole, and 4(β-aminoethyl)-5-hydroxyindole (FAEFHI) in which the side chain is flexible, and connected to the indole at the C4 position (rather than at C3 as in 5-HT). Ab initio molecular orbital calculations of the molecules and model fragments were performed with STO-3G and 3-21G basis sets, using structural optimization procedures. The results show that both structures possess the reactivity elements required for the interaction with the 5-HT receptor, but that FAEFHI cannot be recognized at the 5-HT receptor because the side chain is held in the wrong conformation with respect to the indole portion by a strong hydrogen bond between the side chain amine group and the hydroxyl at C5. We report results from competition experiments for binding at high affinity 5-HT binding sites in brain membranes which support this conclusion by showing that FAEFHI has low affinity for these sites.

Buspirone analogs. 1. Structure-activity relationships in a series of N-aryl- and heteroarylpiperazine derivatives

A series of analogues of buspirone was synthesized in which modifications were made in the aryl moiety, alkylene chain length, and cyclic imide portion of the molecule. These compounds were tested in vitro for their binding affinities to rat brain membrane sites labeled by either the dopamine antagonist [3H]spiperone or the alpha 1-adrenergic antagonist [3H]WB-4101. Compounds were also tested in vivo for tranquilizing properties and induction of catalepsy. Potency at the [3H]spiperone binding site was affected by alkylene chain length and imide portion composition. Nonortho substituents on the aryl moiety had little effect on [3H]spiperone binding affinity. Structure-activity relationships of ortho substituents demonstrated only modest correlations between the receptor binding data and physical parameters of the substituents. The complex nature of the drug-receptor interactions may be understood in terms of the fit of buspirone to a hypothetical model of the dopamine receptor.

Characterization of [3H]desipramine binding associated with neuronal norepinephrine uptake sites in rat brain membranes

A variety of evidence indicates that [3H]desipramine can label neuronal norepinephrine uptake sites in brain membranes. Pretreatment of rat cerebral cortical membranes with 0.3 M KCl increases the ratio of high affinity to low affinity saturable [3H]desipramine binding. With this improved tissue preparation, we have confirmed our earlier observation that the high affinity [3H]desipramine binding component (KD = 2 to 4 nM) is associated with norepinephrine neuronal uptake sites. The potencies of various antidepressant drugs in reducing [3H]desipramine binding correlate with their inhibition of neuronal [3H]norepinephrine accumulation. Like the norepinephrine uptake system, high affinity [3H]desipramine binding is dependent both on sodium and chloride, with half-maximal stimulation by 10 mM chloride. Although bromide can substitute for chloride to stimulate binding, other anions, including iodide, fluoride, acetate, citrate, and phosphate, are inactive. Comparable sodium and anion regulation of [3H]imipramine binding to serotonin uptake recognition sites also is observed. The association of [3H]desipramine binding sites with neuronal norepinephrine uptake sites is supported further by the selective abolition of high affinity [3H]desipramine binding following the destruction of central norepinephrine neurons by intraperitoneal administration of DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine). In vitro incubation of cerebral cortical membranes with DSP-4 also selectively abolishes the high affinity [3H]desipramine binding, an effect which cannot be reversed by repeated washing of the membranes, suggesting that DSP-4 alkylates neuronal norepinephrine uptake sites.

Regulation of neocortical muscarinic receptors: effects of drug treatment and lesions

Regulation of neocortical muscarinic receptors: effects of drug treatment and lesions

Barbiturate receptor sites are coupled to benzodiazepine receptors.

Barbiturates enhance the binding of [3H]diazepam to benzodiazepine receptor sites in rat brain. This effect occurs at pharmacologically relevant concentrations of barbiturates, and the relative activity of a series of compounds correlates highly with anesthetic activity of the barbiturates and with their ability to enhance postsynaptic inhibitory responses to the neurotransmitter gamma-aminobutyric acid. Barbiturate enhancement of benzodiazepine binding is stereospecific, with the more active anesthetic isomers of N1-methylbarbiturates being also more active than their stereoisomers in enhancing benzodiazepine binding. The active barbiturates produce a reversible enhancement in the affinity of specific benzodiazepine binding with no effect on the number of binding sites. The barbiturate enhancement, but not the baseline benzodiazepine binding, is competitively inhibited by the convulsant picrotoxinin (at 1-10 microM), a drug that has been shown to label barbiturate-sensitive brain membrane sites related to the gamma-aminobutyric acid receptor-ionophore complex. The barbiturate effect is also dependent upon the presence of certain anions, and only those anions, that penetrate the chloride channels regulated by gamma-aminobutyric acid receptors. These results suggest that picrotoxin-sensitive barbiturate binding sites are coupled to benzodiazepine receptors in the gamma-aminobutyric acid receptor-ionophore complex, and that these binding sites have the properties of pharmacologically relevant receptors that mediate at least part of the action of various nervous system depressant and excitatory drugs.

Specific binding of 3H-adenosine to rat brain membranes.

The binding of 3H-adenosine to rat brain membranes was studied by a microcentrifugation technique. Specific binding of 3H-adenosine was rapid, reversible, saturable and dependent on pH and temperature. Scatchard plots of equilibrium binding data were nonlinear suggesting the existence of two different binding sites for adenosine. The dissociation constants (Kd) were 1.7 muM and 13.6 muM and the maximal number of binding sites (Bmax) 31 and 165 pmol adenosine bound per mg of membrane protein. Ten adenosine derivatives were studied for their ability to compete with 3H-adenosine binding. The phosphorylated adenosine compounds 5'-AMP, cyclic AMP and ATP were most potent in displacing 3H-adenosine from its binding sites and the IC50-values ranged from 11--25 muM. N6-Phenylisopropyladenosine produced only partial inhibition (30%) of 3H-adenosine binding and no stereospecific difference between the (-)- and (+)isomer was observed. Several methylxanthines known as adenosine antagonists competed for the 3H-adenosine binding sites parallel with their pharmacological potency. The results offer a first approach for the study of adenosine binding sites in brain membranes.

Brain α-adrenergic receptors: comparison of [ 3H]WB 4101 binding with norepinephrine-stimulated cyclic AMP accumulation in rat cerebral cortex

The ability of a series of adrenergic agents to displace the binding to brain membranes of [ 3H]WB 4101, a potent α-adrenergic antagonists (WB4101 = 2[2-(2,6-dimethoxyphenoxy)ethylaminomethyl]-1,4-benzodioxane hydrochloride), has been compared with the potency of these agents in stimulating or inhibiting the α-adrenergic component of cyclic AMP accumulation in rat cerebral cortex slices. [ 3H]WB 4101 rapidly bound to a high affinity site ( K D = 2.7nM) in membranes from cerebral cortex Binding came to equilibrium by 2 min at 37°C and was rapidly reversed in the presence of phentolamine. The potencies of adrenergic agents (WB 4101 > phentolamine > naphazoline) in displacing binding of [ 4H]WB 4101 were comparable to the potencies of these agents as inhibitors of the α-adrenergic component of norepinephrine-stimulated cyclic AMP accumulations. Phenoxybenzamine, clonidine, chlorpromazine and haloperidol were about 10–30 times more potent in inhibiting cyclic AMP accumulation than in displacing [ 3H]WB 4101 binding. The potency of classical α-adrenergic agonists in displacing WB 4101 (epinephrine > norepinephrine > methoxamine) correlated with the ability of these agonists to increase cyclic AMP levels. Overall a significant correlation ( r = 0.87, P < 0.005) was found between WB4101 binding and α-AAadrenergically mediated cyclic AMP accumulation in brain. Several ligands bind to specific sites in brain membranes with α-adrenergic receptor properties. The identificatiom of these binding sites as receptors depends on a correlation of binding with a known α-adrenergic receptor-mediated response in brain. These data demonstrating that WB4101 correlates with norepinephrine-stimulated cyclic AMP accumulation suggest that WB 4101 may bind to the membrane receptor sites mediating the α-adrenergic accumulation of cyclic AMP in rat cerebral cortex.