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.     

Multiple binding sites for [3H]clonidine and [3H]WB-4101 in rat brain

Binding of the alpha-adrenergic agonist [3H]clonidine and the alpha-adrenergic antagonist [3H]WB-4101 exhibited multiple binding site characteristics in both rat frontal cortex and cerebellum. Kinetic analysis of the dissociation of both radioligands in rat frontal cortex suggests two high affinity sites for each ligand. Competition of various noradrenergic agonists and antagonists for [3H]WB-4101 binding yielded shallow competition curves, with Hill coefficients ranging from 0.45 to 0.7. This further suggests multiplicity in [3H]WB-4101 binding. In the rat cerebellum, competition of various noradrenergic drugs for [3H]clonidine binding yielded biphasic competition curves. Furthermore Scatchard analysis of [3H]clonidine binding in rat cerebellum showed two high affinity sites with KD = 0.5 nM and 1.9 nM, respectively. Competition of various noradrenergic drugs for [3H]WB-4101 binding in the rat cerebellum yielded biphasic competition curves. Lesioning of the dorsal bundle with 6-hydroxydopamine did not significantly affect the binding of either [3H]clonidine or [3H]WB-4101. These findings for both [3H]clonidine and [3H]WB-4101 binding in rat frontal cortex and cerebellum can be explained by the existence of postsynaptic binding sites for both 3H ligands.     

Temperature-sensitive high affinity [3H]tryptamine binding sites in rat brain

The influence of prior incubation on [3H]tryptamine binding was investigated in rat brain synaptic plasma membranes. A 55 min preincubation of the membranes at 37 degrees C induced an approx. 2.4-fold increase in the specific binding of [3H]ligand to the subsequently washed preparations and this phenomenon was quite temperature-dependent. On the other hand, the proportion of nonspecific binding sites was significantly decreased by 70% of the original sites within 20 min of the start of preincubation. Pargyline, ascorbic acid, EGTA, metal ions (Ca2+, Mg2+, Na+) and guanine nucleotides, included in the preincubation buffer, were all inactive on the stimulation of [3H]tryptamine binding, while the pretreatment of membranes with glutaraldehyde antagonized the augmentation of this binding. Furthermore, it was revealed that the Scatchard plot of the [3H]tryptamine binding preincubated at 0 degree C conformed to a straight line (KD = 33.1 nM, Bmax = 543 fmoles/mg protein), whereas a curvilinear Scatchard plot was obtained at 37 degrees C preincubation. Nonlinear regression analysis of the latter resulted in apparent KD (nM) & Bmax (fmoles/mg protein) values of 0.45 & 102.7 and 33.7 & 603.4 for the high and low affinity sites, respectively. All these observations lead to the inference that the preincubation-induced increase in [3H]tryptamine binding (i.e., nearly high affinity proportion of sites) may occur as a result of temperature-sensitive interconvertible conformational changes.     

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.     

5-Hydroxytryptamine and (+)-lysergic acid diethylamide displace [3H]thyrotropin-releasing hormone at its binding sites in the rat limbic forebrain

Binding of [3H]thyrotropin-releasing hormone (TRH) to its receptors in the rat limbic forebrain was partially displaced by 5-hydroxytryptamine (5-HT, ligand for 5-HT1 receptors) and (+)-lysergic acid diethylamide ((+)-LSD, ligand for 5-HT1 and 5-HT2 receptors) at nanomolar concentrations. Spiperone (ligand for 5-HT2 receptors) displaced [3H]TRH in a dose-dependent manner at micromolar concentrations. These results suggest that some TRH receptors are related to 5-HT1 receptors, probably adjoining them on the membrane. This type of TRH receptor is shown to be among the high-affinity receptors which we reported previously. The significance of the receptor-coexistence is such that TRH facilitates serotonergic transmission by increasing the density of 5-HT1 receptors. This finding seems to support a pharmacological observation of other investigators that TRH potentiates 5-HT-induced hyperactivity in mice, probably by affecting postsynaptic 5-HT receptors.     

Biochemical characterization of [3H]tryptamine binding sites from rat brain

Rat brain membranes were treated with different protein modifying reagents, all of which were able to reduce [3H]tryptamine binding. However, inactivation by N-ethylmaleimide and iodoacetamide only was counteracted by coincubation with tryptamine. Thus, the [3H]tryptamine binding molecule is a membrane protein with an essential sulfhydryl group at the binding site. After incubation of digitonin-solubilized membranes with seven different lectins, no precipitation of [3H]tryptamine binding sites was observed. On concanavalin A and wheat germ agglutinin affinity chromatography, no [3H]tryptamine binding activity was found to be specifically bound. Therefore, the [3H]tryptamine binding protein appears to be devoid of lectin binding carbohydrate residues.     

Increased specific binding of [3H]diazepam in rat brain following chronic diazepam administration.

Male rats (175 g) were given 30 mg of diazepam in their food daily for 35 days. The animals became drowsy and ataxic from this high dose of drug. After the 35-day dosing, the rats were killed daily, and specific binding of [3H]diazepam and [3H]flunitrazepam was determined in synaptosomal preparations from these and corresponding control rats. On days 3, 4, 6, and 7 after the treatment period the specific binding and specific binding of [3H]diazepam was double that of the control binding and specific binding of [3H]flunitrazepam was 1.67 times that of control. The data indicate that very high doses of diazepam, given for long periods, cause increased specific binding of radiolabeled ligand to brain subfractions. The possible mechanisms and implications are discussed. When lower doses or shorter dosage regimens are used, increased binding is not observed.     

In situ imaging of specific binding of [3H]isatin in rat brain

Isatin is an endogenous indole that influences a range of processes both in vivo and in vitro. It has a distinct and discontinuous distribution in the brain, as well as in other mammalian tissues and body fluids. However, the distribution of isatin binding sites in the brain is not known. Using a real-time beta-imager we have investigated the distribution of [3H]isatin-specific binding in rat brain sections. The highest labeling was found in hypothalamic nuclei and in the cortex, hippocampus, and cerebellum. Administration of the mechanism based monoamine oxidase inhibitor, pargyline, reduced but did not abolish the specific binding of [3H]isatin in the rat brain. The distribution became cortex, cerebellum, hypothalamus > hippocampus > brain stem > thalamus approximately striatum.     

Endogenous inhibitor of [3H]kainate binding to synaptic membrane in rat brain

An understanding of the mechanism of kainic acid toxicity to neurons could provide important clues to pathogenesis of Huntington's chorea. The existence of high-affinity binding sites for kainate, a foreign compound, is suggestive of the existence of kainate-like substances in the brain. In addition to such neurotoxic kainate-like substances, and endogenous inhibitor of kainate binding may also exist in the brain to allow the synaptic function to operate normally. Based on this idea, the existence of molecules which inhibit [3H]kainate binding to synaptic membranes was examined in rat brain. An endogenous inhibitor of [3H]kainate binding to synaptic membranes was found in the supernatant obtained from synaptic membranes of rat brain. The inhibitor is a thermostable, basic protein with a relatively low molecular weight.     

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.     

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

In situ molecular weight determination of rat brain [3H]phenytoin binding sites

Using irradiation inactivation analysis of specific [3H]phenytoin binding to rat brain we have demonstrated that there are two different binding sites involved, with molecular weights of 73238 +/- 1535 (higher affinity site) and 108121 +/- 6935 (lower affinity site).     

Detailed analysis of heterogeneity of [3H]naloxone binding sites in rat brain synaptosomes

The experiments reported in this paper address the question of heterogeneity of [³H]naloxone binding sites in rat brainstem synaptosomal preparations at 23°C in the presence of 100 mM sodium chloride. Kinetic analysis in the presence of 0.4, 4 and 10 nM [³H]naloxone gave pseudo-first order association rate of 0.9±0.04, 1.23±0.08 and 1.06±0.08 min^–1, respectively. The dissociation of a 1 nM [³H]naloxone receptor complex was biphasic with dissociation rate constants of 1.8 and 0.4 min^–1. On the other hand, dissociation of 10 nM [³H]naloxone was monophasic with ak _d of 1.1 min^–1. Two subpopulations of binding sites were also observed by steady state binding studies, with Kd values of 0.5 and 3.4 nM and a ratio of high to low affinity sites of 1:9. Heterogeneity of [³H]naloxone binding sites could be seen by displacement studies performed with opioid eptides and alkaloids. We suggest that our data best fits a model with two independent naloxone binding sites wherein either one or both undergo a multi-step interaction with ligand.     

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.     

Comparison of [3H] phencyclidine ([3H] PCP) and [3H] N-[1-(2-thienyl) cyclohexyl] piperidine ([3H] TCP) binding properties to rat and human brain membranes

The investigation of [3H] PCP and [3H] TCP binding properties to rat cerebrum and cerebellum resulted in the demonstration of multiple binding sites for the two drugs. In the two tissue preparations PCP had a lower affinity than TCP. In membranes from the cerebrum an equal number of high affinity binding sites were present for [3H] PCP and [3H] TCP. However, low affinity binding sites were two times more numerous for [3H] PCP than for [3H] TCP. In the cerebellum, the number of high and low affinity sites labeled by the two radioligands was identical, but the number of high affinity sites was about 7 fold lower than in the cerebrum. Taken together these results may indicate that in the cerebrum [3H] PCP labels other sites than NMDA/PCP receptor(s), maybe sigma receptors and/or the dopamine uptake complex. In human cerebral cortex samples [3H] TCP also bound to two different sites. The number of high and low affinity sites were 12 and 3 times, respectively, less abundant than in the rat cerebrum. Low affinity sites were of higher affinity (5 times) than corresponding sites in the rat brain. In the human cerebellum [3H] TCP binding parameters were identical to those measured in the same region in the rat.     

Calcium alters the properties of [3H]diazepam binding sites in rat brain membranes

• 1.1. [³H]diazepam ([³H]DZ) was used as a ligand to study the effects of Ca²⁺ on benzodiazepine binding to rat brain membranes.
• 2.2. [³H]DZ bound at a single class of binding sites showing K_D and B_max values of 5.4 nM and 852 fmol/mg protein respectively. These values are consistent with previous reports.
• 3.3. Amongst the various divalent cations tested Mg²⁺, Fe²⁺, Cd²⁺, and Sr²⁺ had no significant effect on [³H]DZ binding. Mn²⁺, Ba²⁺, Co²⁺, Ni²⁺ and La²⁺ enhanced radioligand binding, whereas Ca²⁺, Zn²⁺ and Pb²⁺ inhibited [³H]DZ binding to brain freeze-thawed membranes.
• 4.4. The inhibition of [³H]DZ binding by Ca²⁺ was concentration-dependent. 50% inhibition occurred at a Ca²⁺ concentration of 5.6mM. The Hill coefficient for the inhibition was 1.03, displaying noncoperativity. The effect of Ca²⁺ on [³H]DZ binding could be prevented by La³⁺ but was not reversed by EGTA.
• 5.5. A kinetic analysis of Ca²⁺ inhibition of [³H]DZ binding indicates that Ca²⁺ inhibited competitively. Analysis of binding isotherms indicates that both K_D and B_max were altered at the [³H]DZ binding sites. The marked increase in K_D value in the presence of Ca²⁺ (5 mM) can be explained by a drastic increase in the dissociation rate constant.
• 6.6. It was suggested that Ca²⁺ may induce a conformational change in the diazepam binding sites on rat brain membranes. The unchanged Hill coefficient in the presence or absence of Ca²⁺ indicates that a single population of binding sites was labeled by [³H]DZ.
• 7.7. The calmodulin antagonists, trifluoperazine and W-7 were weak inhibitors of [³h]dz binding.

     

Quantitative autoradiography of [3H]CTOP binding to mu opioid receptors in rat brain

[3H]H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ([3H]CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with [3H]CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited [3H]CTOP binding with high affinity (IC50 values of 0.2-2.4 nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69, 593 were very weak inhibitors of [3H]CTOP binding (IC50 values of 234-3631 nM). Light microscopic autoradiography of [3H]CTOP binding sites revealed regions of high density (nucleus of the solitary tract, clusters in the caudate-putamen, interpeduncular nucleus, superior and inferior colliculus, subiculum, substantia nigra zona reticulata, medial geniculate, locus coeruleus and dorsal motor nucleus of the vagus) and regions of moderate labeling (areas outside of clusters in the caudate-putamen, cingulate cortex, claustrum and nucleus accumbens). The cerebral cortex (parietal) showed a low density of [3H]CTOP binding.