Characterization of [3H]clozapine binding sites in rat brain

We examined the characteristics of [3H]clozapine binding sites in four rat brain regions (frontal cortex, limbic area, hippocampus and striatum) in order to elucidate the pharmacological profile of this unique atypical antipsychotic drug. The specific [3H]clozapine binding was found to be saturable and reversible in all these brain regions. Scatchard analysis of the saturation data indicated that the specific binding consisted of high- and low-affinity components. Displacement experiments showed that the muscarinic cholinergic receptor represented about 50% of [3H]clozapine binding in each brain area. Serotonin 5-HT2 and dopamine D4 receptor binding sites could also be detected by displacement experiments using ketanserin and nemonapride, respectively, in frontal cortex and limbic area, but not in hippocampus or striatum. Alpha-1, alpha-2, histamine H1, dopamine D1, D2, or D3 receptor components could not be determined within the high-affinity [3H]clozapine binding sites in any brain region. It is possible that the atypical property of clozapine may depend on the modulatory effect on dopaminergic function via 5-HT2 receptor blockade and/or may be mediated via D4 receptor blockade in the mesocortical and mesolimbic area.     

Autoradiographic study on [3H]-[D-Ala2]-deltorphin-I binding sites in the rat brain

Previous biochemical and pharmacological studies have shown that [D-Ala2]-deltorphin-I (DADTI) has a high affinity and selectivity for delta-opioid receptors. In this study, designed to provide morphological details, the distribution of DADTI binding sites was examined by autoradiography on coronal, sagittal and horizontal frozen sections of adult rat brain. The sections were incubated with tritiated DADTI solution and exposed for 12 weeks to a 3H-sensitive film. DADTI labelling clearly demonstrated selective and high affinity binding sites of delta-opioid type in several brain regions, including olfactory system, neostriatum, nucleus accumbens, and cortical layers I-II and V-VI.     

Multiplicity of [3H]1,3-di-o-tolylguanidine binding sites with low affinity for haloperidol in rat brain

Specific binding of [3H]1,3-di-o-tolylguanidine (DTG) was found not only in synaptic membrane fractions but also in subcellular fractions enriched of microsomes, nuclei and mitochondria/myelins, with different sensitivities to displacement by the antipsychotic haloperidol. The highest binding was detected in microsomal fractions followed by, in order of decreasing binding, fractions enriched in nuclei, synaptic membranes, mitochondria/myelins and homogenates. [3H]DTG binding was completely abolished by prior treatment of the synaptic membranes with a low concentration of Triton X-100. [3H]DTG binding reached a plateau within 30 min of the incubation at 2 degree C, whereas raising the incubation temperature to 30 degrees C resulted in marked shortening of the time required to attain equilibrium, without altering the binding at equilibrium. The binding was inhibited by haloperidol in a concentration-dependent manner over a concentration range of 1 nM to 0.1 mM but with a potency more than 100 times weaker than the value reported in the literature, irrespective of the termination method employed and the external proton concentrations. [3H]DTG binding was markedly displaced by a variety of compounds including sigma ligands, benzomorphan opiates and noncompetitive antagonists at the N-methyl-D-aspartate (NMDA) receptor in synaptic membranes of the cortex, hippocampus and cerebellum. However, sigma ligands such as haloperidol, DTG and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine were more potent in displacing [3H]DTG binding in cortical membranes than in hippocampal and cerebellar membranes, while the potencies of the NMDA antagonists were not significantly different from each other among these 3 different central structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes of sodium-dependent D-[3H]aspartate and [3H]FK506 binding in rat brain

We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependent D-[3H]aspartate and [3H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependent D-[3H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [3H]FK506 binding was significantly lower in only the hippocampus, thalamus and cerebellum. 3[H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependent D-[3H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependent D-[3H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10-25% reduction in sodium-dependent D-[3H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

High affinity [3H]imipramine and [3H]paroxetine binding sites in suicide brains

Specific binding of [3H]imipramine and [3H]paroxetine was simultaneously examined in human brains (frontal cortex, temporal cortex, cingulate cortex, hypothalamus, hippocampus and amygdala) from 11 controls and 11 depressed suicide victims. A single saturable high affinity site was obtained for both radioligands. Age was not related to significant changes in [3H]imipramine and [3H]paroxetine binding parameters, which indicates the stability of the brain serotonergic system with increasing age. A major finding of the present study concerns the existence of a significant decrease in the maximum number (Bmax) of [3H]imipramine binding sites in hippocampus from depressed suicides as compared with the control group, without changes in the binding affinity (Kd). In contrast, when [3H]paroxetine was used as radioligand, no changes in either Bmax or Kd were detected in any of the brain regions studied. These findings suggest that [3H]imipramine may be a better marker than [3H]paroxetine when alterations in the presynaptic serotonergic uptake site are to be detected.     

Modulation of [3H]mazindol binding sites in rat striatum by dopaminergic agents

A 57-year-old male presented with palpitations and dyspnea on exertion. Examination of the peripheral blood and bone marrow showed pancytopenia with marked red cell aplasia. Hypogammaglobulinemia was also recognized. Chest X-ray and CT showed a mass in the anterior mediastinum. A biopsy showed thymoma. Two months after admission, the patient died of sepsis secondary to worsening pancytopenia and hypogammaglobulinemia. Autopsy showed non-invasive spindle cell type thymoma and a marked decrease of hematogenous cells. Review of the literature indicates that pancytopenia associated with thymoma is resistant to all forms of treatment and its prognosis is poor.     

Interaction of thiocolchicoside with [3H]strychnine binding sites in rat spinal cord and brainstem

Radioreceptor binding assays and receptor autoradiography were used to investigate the activity of thiocolchicoside on strychnine-sensitive binding sites in rat brain and spinal cord using [3H]strychnine as a ligand. Thiocolchicoside displaced the binding of [3H]strychnine with an affinity similar to that of unlabeled glycine, and showed a Hill coefficient and proportionality parameter (P) less than unity. The activity of thiocolchicoside toward [3H]strychnine binding sites was confirmed in autoradiographic studies. The results suggest that thiocolchicoside behaves as an allosteric compound acting on the strychnine-sensitive glycine receptor in rat brainstem and spinal cord, and that this may provide a possible mechanism for the myorelaxant activity of this colchicoside derivative, the first clinically useful drug acting on this receptor.     

Further characterization of [3H]-CGS 21680 binding sites in the rat striatum and cortex

1. The putative high affinity binding site for the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N- ethylcarboxamidoadenosine (CGS 21680) in the rat cerebral cortex was characterized by use of a number of selective A1 and A2 adenosine receptor ligands, and compared to the characteristics of the more abundant striatal A2A receptor. 2. The binding of [3H]-CGS 21680 to cortical membranes was performed at pH 5.5, in order to increase the amount of specific binding. 3. Reduction of the pH from 7.4 to 5.5 increased the apparent affinity of the striatal binding side for both agonists and antagonists. The relative order of potencies of both groups of ligands were the same at both pH values, and were consistent with binding to the A2A receptor. There was no observable change in the Bmax, the values being 415 and 446 fmol mg-1 protein at pH 5.5 and 7.4 respectively. 4. The cortical binding site yielded a Bmax value of 117 fmol mg-1 protein. The relative order of potencies of the adenosine receptor ligands observed at this binding site were not the same as those observed in the striatum, exhibiting a profile with both A1 and A2 characteristics. 5. Further characterization of this cortical binding site in the presence of the A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed a more typical A2A profile. This indicated that under the conditions used there were two components of [3H]-CGS 21680 binding, approximately 20% of the A1 receptor and 80% to the A2A receptor. 6. It is concluded that in the cerebral cortex there is a CGS 21680 binding site showing the characteristic properties of the striatal A2A receptor, and no evidence was obtained for the existence of a novelA2A-like binding site.     

Autoradiographic characterization of [3H]inositol (1,4,5) trisphosphate and [3H]inositol (1,3,4,5) tetrakisphosphate binding sites in human brain

Autoradiographic techniques were used to investigate the characteristics of tritiated inositol(1,4,5)trisphosphate ([3H]IP3) and inositol (1,3,4,5) tetrakisphosphate ([3H]IP4) binding to human brain. In brain sections [3H]IP3 exhibited a two-site binding with KD values of 87 nM and 9.3 microM respectively for the higher and lower affinity sites. [3H]IP4 also bound to two sites with KD values of 43 nM and 1.4 microM, respectively. With the conditions fixed in this study, [3H]IP3 and [3H]IP4 autoradiography in the cortex, caudate, hippocampus and cerebellum were performed. The most prominent [3H]IP3 binding among these regions was found in the cerebellum, particularly in the molecular layer. Within the hippocampus, the subiculum and the CA1 region showed much more prominent binding than the other subfields. [3H]IP4, binding was fairly homogeneous in the regions studied, with the exception of a slightly higher binding in the molecular layer of the cerebellum.     

Inhibition of [3H]quinpirole binding by a monoamine oxidase inhibitor in subcellular fractions of rat striatum

[3H]Quinpirole is a dopamine agonist with high affinity for D2-like dopamine receptors. A number of non-dopaminergic compounds, most notably monoamine oxidase inhibitors (MAOIs), inhibit the binding of [3H]quinpirole, but not other D2-like agonists and antagonists, in rat striatal membranes by a mechanism that does not involve the enzymatic activity of MAO. To further characterize this novel interaction, the subcellular distribution of spiperone-displaceable, "D2-like" [3H]quinpirole-labeled sites in rat striatum was assessed and compared with the distribution of MAOI-displaceable [3H]quinpirole binding (MQB). "D2-like" [3H]quinpirole binding exhibited similar nanomolar affinity in the crude synaptosomal (P2), crude microsomal (P3), and ribosomal, post-microsomal (P4) fractions. Total binding activity (fmol bound/fraction) of "D2-like" [3H]quinpirole binding was concentrated in the synaptosomal fraction (P2B). The subcellular distribution of MQB paralleled that of "D2-like" [3H]quinpirole binding. This suggests that "D2-like" [3H]quinpirole binding and MQB occur at a common membrane-bound binding site.     

Characterization of [3H]staurosporine binding in protein kinase C-II purified from rat brain

The type II protein kinase C (PKC-II) densely present in mammalian brain plays functional roles in CNS. We examined the characteristics of [3H]staurosporine binding to PKC-II purified from rat brain, compared to [3H]phorbol 12, 13-dibutyrate (PDBu) binding. In brief, [3H]staurosporine binding increased by phosphatidylserine (PtdSer) in a concentration-dependent manner and the binding was enhanced by Ca2+ and phorbol 12-myristate 13-acetate (PMA). In the presence of Ca2+, PMA and PtdSer, Bmax of these bindings markedly increased, but KD did not change. These characteristics of binding were similar to [3H]PDBu binding to PKC-II. Although [3H]PDBu binding was not affected by protein kinase inhibitors such as staurosporine, H-7, K-252a and K-252b, [3H]staurosporine binding was inhibited by these inhibitors. [3H]staurosporine binding was inhibited by several ATP analogues, but was not by guanine nucleotides. PtdSer-induced increase in [3H]PDBu binding was inhibited by Zn2+, but Zn2+ induced increase in [3H]staurosporine binding as well as PtdSer and/or Ca2+. Staurosporine would thus appear to bind to a domain different from phorbol ester-binding one in PKC, interactions between both domains may regulate kinase activity, and 1 mol staurosporine and 4 mol phorbol ester may bind to 1 mol PKC-II.     

Influence of aging on rolipram-sensitive phosphodiesterase activity and [3H]rolipram binding in the rat brain

Cytolytic T cells were generated in vitro by culturing purified Balb/c CD4+ T cells with irradiated C57Bl/6 (B6) splenocytes plus anti-IL-4 mAb. Matched, noncytotoxic T cells were similarly generated by culturing purified Balb/c CD4+ T cells with irradiated B6 splenocytes plus recombinant murine IL-4. The latter T cells displayed to cytolytic activity, even in lectin-mediated lysis assays, but produced characteristic cytokines upon contact with specific alloantigens. Transfusion of cytolytic T cell populations into Balb/c SCID mice bearing B6 cardiac allografts resulted in acute allograft rejection within 5 to 10 days. Transfusion of noncytolytic T cell populations into Balb/c SCID mice bearing B6 cardiac allografts also resulted in acute allograft rejection within 7 to 10 days. Limiting dilution analysis (LDA) of infiltrating cells recovered from rejected allografts after collagenase digestion demonstrated that the CD4+ T cells retained their cytolytic or noncytolytic functional phenotypes in vivo throughout the rejection process. These data demonstrate that isolated CD4+ T cell populations can promote rapid acute cardiac allograft rejection, and that cytolytic activity is not necessary for this acute rejection response.     

3H]Rilmenidine-labelled imidazoline-receptor binding sites co-localize with [3H]2-(benzofuranyl)-2-imidazoline-labelled imidazoline-receptor binding sites and monoamine oxidase-B in rabbit, but not rat, kidney

The distribution and relative densities of imidazoline-receptor binding sites (I-RBS) and monoamine oxidase (MAO)-A and -B enzyme(s) in rat and rabbit kidney were compared autoradiographically using fixed nanomolar concentrations of [3H]rilmenidine and [3H]2-(benzofuranyl)-2-imidazoline ([3H]2-BFI) to label I-RBS, and [3H]RO41-1049 and [3H]RO19-6327 to label MAO-A and -B isoenzymes, respectively. In rat kidney, high densities of I-RBS labelled by [3H]rilmenidine were observed in the cortex and outer stripe (120-280 fmol/mg tissue), in contrast to low I-RBS densities labelled by [3H]2-BFI (<4 fmol/mg). A relatively high density of [3H]RO41-1049 binding to MAO-A enzyme was present in all regions of the rat kidney (160-210 fmol/mg) compared with a low density of [3H]RO19-6327 binding to MAO-B (< 25 fmol/mg). Comparison of MAO-A and -B distributions with that of [3H]rilmenidine-labelled I-RBS strongly suggests a lack of association in rat kidney. Similarly, the extremely low densities of [3H]2-BFI-labelled I2-RBS in rat kidney contrasts with the density of MAO-A, but is consistent with the low density of MAO-B. Rabbit kidney cortex and outer stripe contained high relative densities of [3H]rilmenidine-labelled I-RBS (200-215 fmol/mg) and [3H]2-BFI-labelled I2-RBS (45-60 fmol/mg) with lower densities in the inner stripe and inner medulla (< or = 100 and 30 fmol/mg respectively). A high density of MAO-A binding was observed in the inner stripe (515 fmol/mg) with lower levels in the cortex and outer stripe (100-240 fmol/mg), while high densities of MAO-B binding were observed in the cortex and outer stripe (290-450 fmol/mg) with lower levels in the inner stripe (65 fmol/mg). The correlation between the localization of [3H]rilmenidine-labelled I-RBS and [3H]RO19-6327-labelled MAO-B in rabbit kidney (r = 0.87, P = 0.057) suggest that [3H]rilmenidine may label a binding site co-existent with MAO-B, but not MAO-A (n.s.), in this tissue, but rilmenidine did not inhibit [3H]RO41-1049 or [3H]RO19-6327 binding. The distribution of [3H]2-BFI-labelled I2-RBS overlapped the combined distributions of both MAO-A and -B isoenzymes, suggesting that [3H]2-BFI may label sites on both enzymes in the rabbit, but [3H]2-BFI binding only correlated with [3H]RO19-6327 (r = 0.84, P = 0.07), not [3H]RO41-1049 binding (n.s.). Moreover, 2-BFI only inhibited [3H]RO19-6327, not [3H]RO41-1049 binding. These data are consistent with reports that I2-RBS are located on MAO-B and allosterically influence the catalytic site. The relationship of [3H]rilmenidine- and [3H]2-BFI-labelled I-RBS and the identity of non-MAO-associated [3H]rilmenidine-labelled I-RBS requires further investigation.     

Characterization and distribution of binding sites for [3H]-SR 141716A, a selective brain (CB1) cannabinoid receptor antagonist, in rodent brain

SR 141716A belongs to a new class of compounds (diarylpyrazole) that inhibits brain cannabinoid receptors (CB1) in vitro and in vivo. The present study showed that [3H]-SR 141716A binds with high affinity (Kd=0.61 +/- 0.06 nM) to a homogenous population of binding sites (Bmax=0.72 +/- 0.05 pmol/mg of protein) in rate whole brain (minus cerebellum) synaptosomes. This specific binding was displaced by known cannabinoid receptor ligands with the following rank order of potency SR 141716A > CP 55,940 > WIN 55212-2 = delta9-THC > anandamide. Apart from anandamide, all these compounds were found to interact competitively with the binding sites labeled by [3H]-SR 141716A. On the other hand, agents lacking affinity for cannabinoid receptors were unable to displace [3H]-SR 141716A from its binding sites (IC50 > 10 microM). In addition, the binding of [3H]-SR 141716A was insensitive to guanyl nucleotides. Regional rat brain distribution of CB1 cannabinoid receptors detected by [3H]-SR 141716A saturation binding and autoradiographic studies, showed that this distribution was very similar to that found for [3H]-CP 55,940. In vivo, the [3H]-SR 141716A binding was displaced by SR 141716A with ED50 values of 0.39 +/- 0.07 and 1.43 +/- 0.29 mg/kg following intraperitoneal and oral administration, respectively. Finally, the [3H]-SR 141716A binding sites remained significantly occupied for at least 12 hr following oral administration of 3 mg/kg SR 141716A. Taken together, these results suggest that SR 141716A in its tritiated form is a useful research tool for labeling brain cannabinoid receptors (CB1) in vitro and in vivo.     

Selective labelling of 5-HT7 receptor recognition sites in rat brain using [3H]5-carboxamidotryptamine

The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT7 receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (+/-)-pindolol (10 microM)-insensitive [3H]5-CT ([3H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 microM) displayed a pharmacological profile similar to the recombinant 5-HT7 receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (+/-)-pindolol (10 microM)-insensitive [3H]5-CT recognition sites also resembled, pharmacologically, the 5-HT7 receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [3H]5-CT binding to residual, possibly, 5-HT1A sites. Competition for this [3H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT7 receptor. Saturation studies also indicated that (+/-)-pindolol (10 microM)/WAY 100635 (100 nM)-insensitive [3H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (Bmax=33.2+/-0.7 fmol mg(-1) protein, pKd=8.78+/-0.05, mean+/-S.E.M., n=3). The development of this 5-HT7 receptor binding assay will aid investigation of the rat native 5-HT7 receptor.