Characterization of [3H]Vesamicol binding in rat brain preparations

The binding of (1)-[³H]vesamicol was characterized in several subcellular fractions and brain regions of the rat. Binding to a lysed P₂ fraction from the rat cerebral cortex reached equilibrium within 4 min at 37°C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P₂ fractions from the cerebral cortex (K_d:21 nM and 980 nM), striatum (K_d:28 nM and 690 nM), and cerebellum (K_d:22 nM and 833 nM). High affinity B_max values were highest in striatum (1.17 pmol/mg protein), followed by cerebellum (0.67 pmol/mg protein), and cerebral cortex (0.38 pmol/mg protein). Low affinity B_max values were highest in cerebellum (5.2 pmol/mg protein), with similar values for cerebral cortex (3.7 pmol/mg protein) and striatum (3.8 pmol/mg protein). High affinity but not low affinity binding in each brain region was stereospecific. Another inhibitor of vesicular ACh-transport also displaced 1-vesamicol binding potently (IC₅₀:17 nM) and efficaciously (over 90%). Both high affinity and low affinity B_max values for [³H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by puriffied synaptosomes, crude membranes and P₂ fractions. Specific binding was not observed in a mitochondria-enriched fraction. Crude membrane preparations of primary, neuron-enriched whole brain cultures also exhibited high (64 nM) and low affinity (1062 nM) [³H]vesamicol binding. Isoosmotic replaement of 0.18 M KCl in the binding-buffer with NaCl had no effect on binding. These results suggest that at least some high affinity [³H]vesamicol binding in rat brain preparations may be associated with synaptic vesicles, some of which may not be cholinergic in origin.     

Interaction of leucine enkephalin with (3H)naloxone binding in rat brain: Evidence for an opioid receptor complex

We recently presented evidence that distinct morphine and enkephalin receptors coexist in an opioid receptor complex (Mol. Pharmacol. 21:548-557, 1982). In this paper, we present data which demonstrate that in the presence of sodium leucine enkephalin noncompetitively inhibits the binding of [3H]naloxone to a crude particulate fraction of rat brain. Since the binding site labeled by [3H]naloxone in the presence of sodium may be an alternate conformation of the morphine receptor, these data provide further evidence that morphine and enkephalin receptors are allosterically coupled.     

Characterization of radioiodinated (-)-ortho-iodovesamicol binding in rat brain preparations

We investigated the binding characteristics of optical isomers of three iodovesamicol analogs to vesicular acetylcholine transporters (VAChT) and to sigma receptors (sigma-1, sigma-2) in rat brains. In competitive inhibition studies, (-)-enantiomers [(-)-ortho-iodovesamicol ((-)-oIV), (-)-meta-iodovesamicol ((-)-mIV), (-)-vesamicol] displayed a higher affinity for VAChT than (+)-enantiomer [(+)-oIV, (+)-mIV, (+)-vesamicol]. (-)-oIV and (-)-mIV showed the same high affinity for VAChT as (-)-vesamicol. For sigma receptors(sigma-1, sigma-2), (-)-oIV (Ki = 62.2 nM (to sigma-1) and 554 nM(to sigma-2)) showed a lower affinity than (-)-mIV (Ki = 4.5 nM (to sigma-1) and 42.9 nM (to sigma-2)). Furthermore, in a saturation binding study, (-)-[125I]-oIV exhibited a Kd of 17.4 +/- 5.1 nM with a maximum number of binding sites, Bmax, of 559 +/- 51 fmol/ mg of protein. These results showed that (-)-oIV binds to vesicular acetylcholine transporters (VAChT) more selectively than (-)-mIV, previously reported as a VAChT mapping agent, and may be suitable for VAChT imaging studies.     

In vivo morphine decreases [3H]nimodipine receptor binding in rat brain regions

Thein vivo effect of the mu agonist morphine and antagonist naloxone on [³H]nimodipine receptor binding in rat brain regions has been investigated. Morphine administration (15 mg/s.c.) for thirty minutes produced a 19% decrease in [³H]nimodipine receptor binding (B _max 158.2 fmol to 128.9 fmol) in cortex and 29% decrease in cerebellum (65.3 fmol to 46.0 fmol). Lesser changes were observed in hippocampal and striatal regions with no changes in hypothalamus and brain stem. All effects were completely antagonized by naloxone pretreatment (1 mg/kg). The studies suggest that opiates in vivo can alter [³H]nimodipine binding to the Ca²⁺ channel receptor protein. These findings agree with the previously observed decreases in Ca²⁺ influx in nerve ending preparations and inhibition of I_Ca ²⁺ following opiate treatment and suggest opiates reduce Ca²⁺-dependent neurotransmitter release by altering the Ca²⁺ channel receptor protein in an allosteric fashion.     

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Characterization of 3H-AVP binding sites in particulate preparations of rat brain

Specific binding sites for vasopressin (AVP) were located in subcellular particulate fractions of rat brain with tritiated vasopressin of high specific activity, 22.5 Ci/mmol. Rat brain tissue was dissected, placed in cold 0.32 M sucrose containing proteolytic inhibitors, homogenized and fractionated into a crude nuclear fraction (1K pellet), crude mitochondrial fractions (12K pellet), and plasma membranes and microsomes (100K pellet). Specific binding of vasopressin was found in the 12K and 100K pellets in the presence of a divalent metal ion with Ni greater than Co greater than Mg greater than Mn greater than no metal ion at pH 7.4 in 50 mM Tris-Maleate buffer. Maximum specific binding of 16 nM AVP was located in the 100K anterior cortex fraction which bound 350 fmoles/mg protein; striatum, midbrain/thalamus, cerebellum, and medulla oblongata and pons bound specifically about 200 fmoles/mg protein and frontal poles and parietal cortex about 100 fmoles/mg protein in the 100K pellet. In all of the brain regions studied, except hippocampus and septum, the 100K pellet bound specifically 2 to 4 times more 3H-AVP than the 12K pellet. In the hippocampus with 16 nM AVP, the 12K pellet bound specifically 150 fmoles/mg protein; the septum, 75 fmoles/mg protein. Little or no binding to the 100K pellet was present in these regions. Bound AVP could be dissociated rapidly from the membranes by the addition of EDTA. The 12K hippocampal pellet was further fractionated into myelin, mitochondria, and synaptosomes; purification was confirmed by marker enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kainate binding to the AMPA receptor in rat brain

Displacement of [³H]AMPA and [³H]CNQX by kainate was measured in membranes and solubilized fractions from rat brain. In soluble fractions, plots of [³H]AMPA and [³H]CNQX binding displaced by kainate resulted in one-site fits with K_i values in the range of 1–3 M. In membranes, plots of [³H]AMPA binding displaced by kainate resulted in graphs which were better fit by twosite regression analysis than by a one-site fit. The K_i value for the high-affinity component of these two-site fits was 3–9 M and the low-affinity component K_i was in the range of 70–120 M; similar values were determined for kainate displacement of [³H]CNQX. The presence of thiocyanate ions had no effect on kainate displacement of [³H]CNQX. Since the affinity for kainate of the presumed synaptic AMPA receptor is in the range of EC₅₀ values for kainate determined from physiological studies, these data contribute further evidence for the idea that kainate binding to synaptic AMPA receptors may be responsible for many of kainate's physiological effects.     

Peptide E: opiate receptor binding profile in the rat brain membrane assay. Comparison with beta-endorphin

Beta-endorphin (beta-EP) and peptide E were compared in respect to their binding potency in the rat brain membrane by radioreceptor binding assay using tritiated human beta-EP, [D-Ala2,D-Leu5]-enkephalin (DADLE), dihydromorphine (DHM) and ethylketocyclazocine (EKC) as primary ligands. When the potency of beta h-EP was chosen to be 100%, peptide E was equipotent with beta-EP in displacing DHM (95%) and EKC (103%) less potent for competing with beta h-EP (60%) and least active (7%) for displacing DADLE. It may be concluded that peptide E binds preferentially with the opiate mu and kappa receptors in the rat brain membrane.     

In vitro autoradiographic localization of (125)i-secretin receptor binding sites in rat brain

Although the existence of the receptor for secretin in the brain was suggested, the localization of secretin receptor and the neuronal function of secretin have not been clarified yet. In the present study, the localization of secretin receptor was investigated in the rat brain by using an in vitro autoradiography technique. Frozen section autoradiography with (125)I-secretin showed intense binding in the nucleus of solitary tract, laterodorsal thalamic nucleus, and accumbens nucleus; moderate binding in the hippocampus, caudate/putamen, cerebellum, cingulate and orbital cortices. Scatchard plot analysis gave the Kd value of 125 pM with Bmax of 134 fmol/mg tissue in the hippocampus. The binding specificity was confirmed with secretin and its analogs, VIP, PACAP, and glucagon. These results indicate the secretin receptor system might have some neural functions in the brain, which could give the basis for therapeutic use of secretin in autistic children.     

High affinity (3H)tryptamine binding sites in various organs of the rat

(3H)Tryptamine binds with high affinity (KD values around 4 nmol/l) to membranes from different organs of the rat. The number of binding sites was high in the liver, intermediate in the heart and cerebral cortex and low in other organs including ileum, kidney, spleen, stomach and lung. Binding to kidney membranes was investigated in more detail. It was rapid and reversible (t1/2 7 min.). The structure-activity profile was characterized by high affinity of some beta-carbolines (IC50 = 20 - 200 nmol/l), medium affinity of serotonin (IC50 = 858 nmol/l) and low of methysergide (IC50 greater than 20 mumol/l). The findings correlate very well with binding characteristics to cerebral cortex membranes. Therefore, rat brain cortex and other organs contain the same type of (3H)tryptamine binding site and may be equally appropriate for further analysis.     

Opiate receptor binding in the brain of rat during stress

Two types of opioid receptors were studied in the brain of rats: Delta (for endogenous opiate) and mu (for exogenous opiates). 3H derivates: D-Ala2-enkephalin and Naloxone were used as labeled ligands. The results obtained were calculated by computer program for automatic estimation of the data using approximation equations. An increase of binding delta receptors is observed in both types of stress (2-8 times), while to the mu receptors the binding is less effective mainly after irradiation. These data suggest that a close interaction exists between sympathoadrenal system and opioid mechanisms during stress.     

Different subtypes of tachykinin NK(1) receptor binding sites are present in the rat brain

(2-[(125)I]iodohistidyl(1))Neurokinin A ([(125)I]NKA), which labels "septide-sensitive" but not classic NK(1) binding sites in peripheral tissues, was used to determine whether septide-sensitive binding sites are also present in the rat brain. Binding studies were performed in the presence of SR 48968 (NK(2) antagonist) and senktide (NK(3) agonist) because [(125)I]NKA also labels peripheral NK(2) binding sites and, as shown in this study, central NK(3) binding sites. [(125)I]NKA was found to label not only septide-sensitive binding sites but also a new subtype of NK(1) binding site distinct from classic NK(1) binding sites. Both subtypes of [(125)I]NKA binding sites were sensitive to tachykinin NK(1) antagonists and agonists but also to the endogenous tachykinins NKA, neuropeptide K (NPK), and neuropeptide gamma (NPgamma). However, compounds of the septide family such as substance P(6-11) [SP(6-11)] and propionyl-[Met(O(2))(11)]SP(7-11) and some NK(1) antagonists, GR 82334, RP 67580, and CP 96345, had a much lower affinity for the new NK(1)-sensitive sites than for the septide-sensitive sites. The hypothalamus and colliculi possess only this new subtype of NK(1) site, whereas both types of [(125)I]NKA binding sites were found in the amygdala and some other brain structures. These results not only explain the central effects of septide or SP(6-11), but also those of NKA, NPK, and NPgamma, which can be selectively blocked by NK(1) receptor antagonists.