Rat Brain Membrane Preparations Research Articles

Cyclic somatostatin octapeptide analogues with high affinity and selectivity toward mu opioid receptors

A series of cyclic conformationally restricted penicillamine containing somatostatin octapeptide analogues have been prepared by standard solid phase synthetic techniques and tested for their ability to inhibit specific [ 125I]CGP 23,996 (des-Ala 1-,Gly 2-[desamino-Cys 3Tyr 11]-dicarba 3,14- somatostatin), [ 3H]naloxone or [ 3H]DPDPE ([D-Pen 2-D-Pen 5]enkephalin) binding in rat brain membrane preparations. We now report structure-activity relationship studies with the syntheis of our most potent and selective mu opioid receptor compound DPheCysTyrDTrpOrnThrPenThrNH 2, which we refer to as Cys 2Tyr 3Orn 5Pen 7-amide. While this octapeptide exhibited high affinity (IC 50 = 2.80 nM) for an apparently single population of binding sites (n H = 0.89 ± 0.1) and exceptional selectivity for mu opioid receptos with an IC 51(DPDPE)/IC 50 (naloxone) ratio of 4,829, it also displayed very low affinity for somatostatin receptors (IC 50 = 22,700 nM). Thus, Cys 2Tyr 3Orn 5 Pen 7-amide may be the ligand of choice for further characterization of mu opioid receptors and for examining the physiological role of this class of receptors.

A new photoaffinity probe, 4-amino-2-[4-(4-azidocinnamoyl)piperazino]-6,7-dimethoxyquinazoline, for α1-adrenoceptors

A photoaffinity probe for α 1-adrenoceptors was synthesized and its properties examined on rat brain membrane preparations. The binding of 4-amino-2-[4-(4-azidocinnamoyl)piperazino]-6,7-dimethoxyquinazoline (ACP) to these receptors was of high affinity (K D = 1.05 nM) and reversible in the dark. A dose-dependent decrease in the concentration of [ 3H]prazosin binding sites without a change in K D was observed when membranes were preincubated with ACP, photolyzed, and then extensively washed prior to assay. This reduction in receptor concentration was prevented by α 1-adrenergic ligands. The specificity of ACP for α 1-receptors was further demonstrated by its inability to compete with [ 3H]dihydroalprenolol and [ 3H]yohimbine binding in these same membranes. Also, the concentrations and affinity constants of β-adrenoceptors and α 2-adrenoceptors were unaffected in membranes which had been photolyzed after preincubation with ACP. No reduction in concentration of α 1-adrenoceptors was detected if ACP was photolyzed prior to incubation with receptors or if ACP was maintained in darkness throughout the experiment. The results suggest that ACP is a specific and sensitive photoprobe that may be useful for further studies on α 1-adrenoceptor coupled systems and that may be particularly suited for use in cell culture work.

Enhancement of brain calcium antagonist binding by phencyclidine and related compounds.

The abilities of compounds structurally or pharmacologically related to phencyclidine to increase the apparent affinity of the [3H]dihydropyridine calcium channel antagonist [3H]nitrendipine were examined in lysed synaptosomal membrane preparations of rat brain. The p-bromo analog of phencyclidine (1-(1-(4-bromophenyl)cyclohexyl)piperidine) was the most efficacious compound tested in enhancing the apparent affinity of [3H]nitrendipine. The efficacy of this compound was approximately two-fold greater than PCP. The stereoisomers of PCMP (1-(1-phenylcyclohexyl-3-methylpiperidine) were also more efficacious than phencyclidine, although only a small degree of stereoselectivity was observed. Levoxadrol, dexoxadrol and the enantiomers of ketamine did not potentiate [3H]nitrendipine binding. The enantiomers of SKF 10047 (n-allylormetazocine), dextrorphan, levorphanol and the ion channel toxins histrionicotoxin and pumiliotoxin-B also increased the apparent affinity of [3H]nitrendipine, while several local anesthetics and mu-opiate receptor ligands were without effect. These studies suggest that the ability of phencyclidine and structurally related compounds to increase the apparent affinity of [3H]nitrendipine is not mediated through an interaction with phencyclidine receptors, but may represent a unique site for allosteric modulation of neuronal dihydropyridine calcium channel antagonist binding sites.

Interactions of divalent cations with single calcium channels from rat brain synaptosomes.

Voltage-dependent calcium channels from a rat brain membrane preparation ("synaptosomes") were incorporated into planar lipid bilayers. The effects of calcium, barium, strontium, manganese, and cadmium ions on the amplitudes and kinetics of single channel currents were examined. The order of single channel conductances was gBa greater than gSr greater than gMn, which was the inverse of the order of the mean channel open times: TMn greater than TCa = TSr greater than TBa. In contrast, the identity of the charge carrier had little or no effect on the mean closed times of the channel. Manganese, in the absence of other permeant ions, can pass through single channels (gMn = 4 pS). However, when added to a solution that contained another type of permeant divalent cation, manganese reduced the single channel current in a voltage-dependent manner. Cadmium, a potent blocker of macroscopic "ensemble" calcium currents in many preparations, reduced the current through an open channel in a manner consistent with Cd ions both not being measurably permeant and interacting with a single site. The permeant ions competed with cadmium for this site with the following order: Mn greater than Sr = Ca greater than Ba. These results are consistent with the existence of no less than one divalent cation binding site in the channel that regulates ion permeation.

Neuropeptide Y receptors in rat brain: Autoradiographic localization

Neuropeptide Y (NPY) receptor binding sites have been characterized in rat brain using both membrane preparations and receptor autoradiography. Radiolabelled NPY binds with high affinity and specificity to an apparent single class of sites in rat brain membrane preparations. The ligand selectivity pattern reveals strong similarities between central and peripheral NPY receptors. NPY receptors are discretely distributed in rat brain with high densities found in the olfactory bulb, superficial layers of the cortex, ventral hippocampus, lateral septum, various thalamic nuclei and area postrema. The presence of high densities of NPY and NPY receptors in such areas suggests that NPY could serve important functions as a major neurotransmitter/neuromodulator in the central nervous system.

3H]Kainic acid binding sites in the synaptosomal-mitochondrial (P2) fraction from goldfish brain

Binding of [ 3H]kainic acid to the synaptosomal-mitochondrial fraction (P2) of the goldfish brain was studied. Specific binding to this fraction represents about half of the total binding capability of the homogenate particulate material and is enriched in synaptic membranes; it is greater by about two orders of magnitude than those given for rat brain and pigeon optic tectum membranes. Association of the ligand-site complex has a time constant lower than 1 min and the same is true for the main component of the dissociation process. The binding equilibrium is apparently not affected by substance contained in the fraction material. The analysis of the dose- response data showed a main receptor population (B max= 139pmol/mg protein) which displayed posperativity (n H = 1.29). The same behaviour was shown by washed membranes from the same fraction but, in this case, the affinity for the ligand was lower (apparent affinity constants: K′ D = 0.28nM for the intact fraction and K′ D = 0.38nM for membranes). A smaller population of sites with higher affinity was also detected both in the intact fraction and in membranes. Among the substances tested as displacers of kainic acid from the synaptosomal sites, the most effective were quisqualate and l-glutamate. Folic acid and its dihydro and tetrahydro derivatives were half as potent as glutamate whereas methyltetrahydrofolic acid and folinic acid had a very weak action. The difference between these sites and those detected on rat brain membrane preparations is discussed.

ChemInform Abstract: OPIOID AGONISTS AND ANTAGONISTS. 6‐DESOXY‐6‐SUBSTITUTED LACTONE, EPOXIDE, AND GLYCIDATE ESTER DERIVATIVES OF NALTREXONE AND OXYMORPHONE

AbstractVerbindungen wie (II)‐(V) werden dargestellt.

High- and low-affinity binding of [ 3H]acetylcholine at nicotinic cholinergic receptors in rat brain

There is both high-affinity and low-affinity nicotinic cholinergic binding of [ 3H]acetylcholine ([ 3H]ACh) in rat brain membrane preparations. As determined by a filtration binding assay, [ 3H]ACh bound with K d =36.0±8.4 nM and B max = 19.4±4.5 fmol/mg protein or 3.3±0.7 fmol/mg tissue for high-affinity binding and K d about 10 −7 to 10 −6 M and B max about 6–10 fmol/mg tissue or 40–60 fmol/mg protein for low-affinity binding. d-Tubocurarine (1 mM) inhibits high- as well as low-affinity binding, whereas 10 μM α-bungarotoxin does not compete at both binding sites. Substance P had no effect on the binding parameters of high-affinity nicotinic cholinergic binding.

Opioid agonists and antagonists. 6-Desoxy-6-substituted lactone, epoxide, and glycidate ester derivatives of naltrexone and oxymorphone

Synthesis and opioid radioreceptor assay data on analogues closely related to 6-desoxy-6-spiro-alpha-methylene-gamma-lactone 5a, a compound with irreversible activity in this assay, are reported. Saturated lactones (7a,b), endocyclic alpha, beta-unsaturated gamma-lactones (8a,b and 9a), and 6 alpha,7 alpha-fused alpha-methylene-gamma-lactones (10a and 11a) were prepared. Related 6-desoxy-6-methylene 6 beta- and 6 alpha-oxides (12a,b and 13a) and glycidate esters 14a,b and 15a,b were also prepared with use of naltrexone (1a) and oxymorphone (1b) as starting material. Compounds in the N-cyclopropylmethyl (N-CPM) series were more potent than those in the N-Me series in displacing [3H]naltrexone in the opioid radioreceptor assay, usually by 2-16-fold in the absence of Na ion. The most potent N-CPM analogues were epoxides 12a and 13a and glycidate esters 14a and 15a, showing IC50's of 2-6 nM, similar to that of 5a. Of the N-Me analogues, 6 beta-oxide 12b was most active, with an IC50 of 8 nM in the absence of Na ion. For the N-CPM analogues, the Na ion ratios were generally less than 1, with two exceptions. The N-Me analogues showed expected larger Na ion effects of 7 or greater. None of the lactone analogues had irreversible effects when preincubated in the rat brain membrane preparation, even at 37 degrees C for 30 min, i.e., washing restored [3H]naltrexone binding to control levels. These results clearly show that the alpha-methylene-gamma-lactone moiety of 5a is required for irreversible effects, consistent with it serving as a conjugate addition acceptor of a nucleophilic group from a ligand at or near the receptor. The epoxides and glycidate esters also had no irreversible activity, indicating more electrophilic functional groups are needed and/or these electrophiles are not properly aligned to react with nucleophilic groups at or near the opioid receptor.

Antagonistic effect of δ-aminovaleric acid on bicuculline-insensitive γ-aminobutyric acid B (GABA B) sites in the rat's brain

The effect of δ-aminovaleric acid (δ-AV) on bicuculline-insensitive γ-aminobutyric acid B (GABA B) sites in the central nervous system (CNS) was investigated by binding studies and experiments on slices in vitro. δ-AV inhibited [ 3H]GABA (10 nM) binding to GABA B sites in a rat brain membrane preparation with an IC 50 value of 10 −4 M. It also inhibited [ 3H]baclofen (20 nM) binding with an IC 50 value of 10 −4 M. In preparations of hippocampal slices, (—)-baclofen (5 μM) reduced the population spikes evoked by stimulating the Schaffer collaterals in CA1 pyramidal cells in the presence of 100 μM bicuculline. δ-AV (1 mM) antagonized this inhibitory action of baclofen. Since baclofen is an agonist of GABA B sites, our results indicate that δ-AV has an antagonistic effect on GABA B sites in the CNS.

Binding characteristics of the bungarotoxin fraction II-S1 to rat brain membranes

Bungarotoxin fraction II-S1 (designated BGT II-S1), isolated from the venom of Bungarus multicinctus, appears to affect nicotinic transmission in rat sympathetic ganglia through its phospholipase activity. On the other hand, the present investigation suggests that other modes of interaction of this toxin with nervous tissue may also exist as, in a rat brain membrane preparation, binding of this toxin to a specific binding site can be demonstrated. In a buffer containing calcium, binding of [ 125I]BGT II-S1 saturated with a B max of approximately 16 fmol/mg protein and a K d of 5 nM. This site did not appear to be directly linked to the nicotinic acetylcholine recognition site as the binding was not displaced by nicotinic agents; however, α-bungarotoxin, which interacts with a nicotinic-like site in neural tissue, did affect the binding and, conversely, BGT II-S1 inhibited the binding of [ 125I]α-bungarotoxin.

Characterization of nitrobenzylthioinosine binding to nucleoside transport sites selective for adenosine in rat brain

Nucleoside transport sites in rat brain membrane preparations were labeled with [3H]nitrobenzylthioinosine ([3H] NBI), a potent inhibitor of nucleoside transport systems. The membranes contained a single class of very high affinity binding sites with KD and Bmax values of 0.06 nM and 147 fmol/mg of protein, respectively. The displacement of [3H]NBI binding by various nucleosides, adenosine receptor agonists and antagonists, and known nucleoside transport inhibitors was examined. The Ki values (micromolar concentration) of [3H]NBI displacement by the nucleosides tested were: adenosine, 3.0; inosine, 160; thymidine, 240; uridine, 390; guanosine, 460; and cytidine, 1000. These nucleosides displayed parallel displacement curves indicating their interaction with a common site labeled by [3H]NBI. The nucleobases, hypoxanthine and adenine, exhibited Ki values of 220 and 3640 microM, respectively. Adenosine receptor agonists exhibited moderate affinities for the [3H]NBI site, whereas the adenosine receptor antagonists, caffeine, theophylline, and enprofylline, were ineffective displacers. The Ki values for cyclohexyladenosine, (+)- and (-)-phenylisopropyladenosine, 2-chloroadenosine, and adenosine 5'-ethylcarboxamide were 0.8, 0.9, 2.6, 12, and 54 microM, respectively. These affinities and the rank order of potencies indicate that [3H]NBI does not label any known class of adenosine receptors (i.e., A1, A2, and P). The Ki values of other nucleoside transport inhibitors were: nitrobenzylthioguanosine, 0.05 nM; dipyridamole, 16 nM; papaverine, 3 microM; and 2'-deoxyadenosine, 22 microM. These results indicate that [3H]NBI binds to a nucleoside transporter in brain which specifically recognizes adenosine as its preferred endogenous substrate. This ligand may aid in the identification of CNS neural systems that selectively accumulate adenosine and thereby control "adenosinergic" function.

Adenosine uptake sites in rat brain: Identification using [ 3H]nitrobenzylthioinosine and co-localization with adenosine deaminase

The binding characteristics of [ 3H]nitrobenzylthioinosine ([ 3H]NBI) to rat brain membrane preparations was examined, and the autoradiographic distribution of this ligand in brain sections was compared with the immunohistochemical localization of adenosine deaminase (ADA). It was found that [ 3H]NBI labels sites for which adenosine has far higher affinity than do other nucleosides, that these sites are heterogeneously distributed and that there is an exact correspondence between areas containing [ 3H]NBI sites and ADA-immunoreactive neurons. Our results indicate that [ 3H]NBI and ADA are potential markers for revealing anatomical sites at which actions of adenosine may be expressed.

Variability of chronic antidepressant treatments on beta-adrenergic receptor sites

1. It has been proposed that down-regulation of beta-adrenergic receptors may be related to the therapeutic action of antidepressants. 2. To further characterize the role of beta-adrenergic receptor regulation in the actions of antidepressants, the effects of chronic treatment with desipramine, zimelidine and bupropion on beta-adrenergic receptors in rat brain membrane preparations were studied. 3. Chronic treatment with desipramine, but not zimelidine and bupropion decreased significantly the density of beta-adrenergic receptors. 4. These data suggest that clinically effective antidepressants may vary considerably in their ability to down-regulate beta-adrenergic receptors after chronic treatment.

Kynurenine may directly interact with GABA receptors in rat brain.

Previous studies have shown that kynurenine may have convulsant activity. In the present investigation the intracerebroventricular injection of L- but not D-kynurenine induced convulsions in the rat. In vitro, L- but not D-kynurenine was able to displace 3H-GABA from rat brain membrane preparations. The action was specific for 3H-GABA and was not observed with other ligands. The data suggest that the convulsant activity of L-kynurenine might be due to an interaction with GABA receptors.

Binding characteristics of dermorphin, and [dermorphin 1–7]- βc-endorphin in rat brain membranes

Dermorphin and a camel beta-endorphin ( β c-EP) analog in which residues 1–7 correspond to the dermorphin sequence ([Dermorphin 1–7]- β c-EP) have been investigated with respect to their receptor binding characteristics using human and camel β-EP as reference peptides. Tritiated dihydromorphine, [D-Ala 2, D-Leu 5]-enkephalin, ethylketocyclazocine and human β-endorphin were used as primary ligands in the rat brain membrane preparation for radioreceptor assay. Camel β-endorphin was the most potent peptide in all experiments. [Dermorphin 1–7]- β c-EP is significantly less potent towards 3H-ethylketocyclazocine and 3H-[D-Ala 2, D-Leu 5]-enkephalin but is as potent towards 3H-dihydromorphine and 3H-human β-endorphin. Dermorphin itself weakly displaces tritiated dihydromorphine, [D-Ala 2, D-Leu 5]-enkephalin and ethylketocyclazocine (potency relative to camel β-EP, 1–4%) but it is more potent (9%) in competition with tritiated human β-endorphin. Dermorphin and the [Dermorphin 1–7]- β c-EP appear to interact preferentially with μ opiate receptors.

ChemInform Abstract: A PHOTOAFFINITY REAGENT TO LABEL THE OPIATE RECEPTORS OF GUINEA PIG ILEUM AND MOUSE VAS DEFERENS

AbstractDargestellt wird das aus einem Peptid und einem Chromophor bestehende Reagens (I), das unter Belichtung irreversibel Opiat‐Rezeptoren blockiert.

Probes for narcotic receptor mediated phenomena. 10. Irreversible ligands to opioid receptors based on biologically potent endoethenooripavines. Reversible binding of fit to mu and delta opioid receptors

A p -isothiocyanatophenylacetylamino moiety at the C7alpha position on an endoethenooripavine (NIH 10358) was found to irreversibly bind to both the mu and delta opioid receptors in rat brain membrane preparations. However, the same endoethenooripavine nucleus with a p -methylfumaroylaminophenylacetylamino moiety at that position (NIH 10364) binds specifically and irreversibly to only mu opioid receptors. Hypotheses are being developed concerning receptor subclass alkylation specificity. For example, it has now been found that the delta receptor irreversible agonist, FIT, can interact with both mu and delta receptors in a reversible manner.

Properties of the specific binding site for arginine vasopressin in rat hippocampal synaptic membranes.

In a previous paper (Pearlmutter, A. F., Constantini, M. G., and Loeser, B. (1983) Peptides 4, 335-341), we have shown that saturable, high-affinity binding sites for [3H]arginine vasopressin (AVP) are located in rat brain membrane preparations. Binding was dependent upon the presence of Ni2+ and could be dissociated by EDTA. In the hippocampus, [3H]AVP binding could be localized to synaptic membranes. In this paper, we characterize in more depth the specificity of [3H]AVP binding to a crude hippocampal synaptic preparation and the metabolism of [3H]AVP in our synaptic preparation. By means of HPLC analysis we demonstrate that the radioactive material specifically bound to hippocampal synaptic membranes is intact [3H]AVP. The ability of analogues of AVP to displace the high-affinity, specific binding of [3H]AVP parallels closely the potency of these analogues to inhibit the extinction of avoidance behavior. In the presence of membrane and Ni2+, [3H]AVP has a half-life of 7 h. In the absence of Ni2+, the half-life of [3H]AVP is 1.2 h. Fractionation by high-pressure liquid chromatography of the supernatant from the incubation media not containing Ni2+ yields three peaks of radioactivity. Analysis of the biological activity of the [3H]AVP peak and the two non-AVP peaks which represent breakdown products show the following: (a) the [3H]AVP peak (52%) and peak III (8%) bind to fresh membranes and (b) peak II (40%) has no binding activity. Although Ni2+, Co2+, benzamidine, and phenanthroline can prevent [3H]AVP degradation, only Ni2+ and, to a much lesser extent, Co2+, can potentiate specific [3H]AVP binding. The results show that AVP-specific binding has properties which parallel its biological activity in behavioral assays; that, ultimately, proteolysis by membrane-bound peptidases inactivates AVP; and that Ni2+ acts both by preventing AVP breakdown and by potentiating specific binding.

A photoaffinity reagent to label the opiate receptors of guinea pig ileum and mouse vas deferens.

An enkephalin derivative, [D-Ala2,Leu5]enkephalin N-[(2-nitro-4-azidophenyl)amino]ethylamide, has been synthesized as a photoaffinity label for the opiate receptor. This compound retains the full biological activity of [D-Ala2,Leu5]enkephalin in guinea pig ileum and mouse vas deferens tests with IC50 values of 4.4 and 2.6 nM, respectively, and inhibits the binding of [3H]naloxone to rat brain membrane preparation with an IC50 value of 2.5 nM. Photolysis of a muscle strip of the guinea pig ileum or of the mouse vas deferens in the presence of the peptide derivative caused irreversible inhibition of electrically stimulated contractions with high efficiencies (80 and 66%, respectively), while the inhibitory effect in the dark was fully reversed by washing. This irreversible inhibition during photolysis was completely prevented by the presence of [D-Ala2,Leu5]enkephalin. These results demonstrate that [D-Ala2,Leu5]-enkephalin N-[(2-nitro-4-azidophenyl)amino]ethylamide is a prominent candidate as a photoaffinity label for the opiate receptor.