Biotinylated-spiperone ligands for quantum dot labeling of the dopamine D2 receptor in live cell cultures

We have synthesized 3 analogs of the dopamine D2 receptor (D2 DR) antagonist spiperone that can be conjugated to streptavidin-coated quantum dots via a pegylated biotin derivative. Using fluorescent imaging we demonstrate that substitution on the spiro position is tolerated, whilst the length and rigidity of a spacer arm attached to spiperone is important in controlling specific labeling as well as minimizing nonspecific labeling to cells and the surface of cell culture dishes. The ligand with the most rigid linker IDT772 (4) had the best binding profile and had high specific binding to D2 DR expressing HEK-293T cells with low nonspecific binding to plates and HEK-293T cells that lacked the D2 DR.     

Use of Cholate/Sodium Chloride for Solubilisation of Brain D2 Dopamine Receptors

The use of cholate (in the presence of sodium chloride) is described for solubilising brain D2 dopamine receptors. The method described gives in high yield a preparation of solubilised D2 receptors which when assayed by [3H]spiperone binding show little interference from nonspecific and nonstereospecific binding sites. Characterisation by ultrafiltration, electron microscopy, gel filtration, and sucrose density gradient centrifugation has been achieved, showing the receptors to be truly solubilised. Pharmacological characterisation using [3H]spiperone binding suggested the presence of serotonergic S2 receptors in addition to D2 receptors. The pharmacological properties of the solubilised D2 receptors show a good correlation with those of membrane-bound receptors, although ligand binding affinities are lower in the solubilised preparation.     

Optimal Conditions for [3H]Apomorphine Binding and Anomalous Equilibrium Binding of [3H]Apomorphine and [3H]Spiperone to Rat Striatal Membranes: Involvement of Surface Phenomena Versus Multiple Binding Sites

I. Binding of [³H]apomorphine to dopaminergic receptors in rat striatum was most reproducible and clearly detectable when incubations were run at 25°C in Tris-HCl buffer, pH 7.5, containing 1 mM-EDTA and 0.01% ascorbic acid, using a washed total-membrane fraction. The receptor binding was stereospecifically inhibited by (+)-butaclamol, and dopamine agonists and antagonists showed high binding affinity for these sites. Unlabelled apomorphine inhibited an additional nonstereospecific binding site, which was unrelated to dopamine receptors. EDTA in the incubation mixture considerably lowered nonstereospecific [³H]apomorphine binding, apparently by preventing the complexation of the catechol moiety with metal ions which were demonstrated in membrane preparations. Stereospecific [³H]apomorphine binding was not detectable in the frontal cortex, whereas in the absence of EDTA much saturable nonstereospecific binding occurred. II. Kinetic patterns of stereospecific [³H]spiperone and [³H] apomorphine binding to rat striatal membranes and the inhibition patterns of a dopamine antagonist and an agonist were evaluated at different temperatures in high-ionic-strength Tris buffer with salts added and low-ionic-strength Tris buffer with EDTA. Apparent K_D, values of spiperone decreased with decreasing tissue concentrations. K_D, values of both spiperone and apomorphine were little influenced by temperature changes. Scatchard plots of the stereospecific binding changed from linear to curved; the amount of nonstereospecific binding of the ³H ligands varied considerably, but in opposite directions for spiperone and apomorphine in the different buffers. In various assay conditions, interactions between agonists, and between antagonists, appeared fully competitive, but agonist-antagonist interactions were of mixed type. The anomalous binding patterns are interpreted in terms of surface phenomena occurring upon reactions of a ligand with complex physicochemical properties and nonsolubilized sites on membranes suspended in a buffered aqueous solution. It is concluded that anomalous binding patterns are not necessarily an indication of binding to multiple sites or involvement of distinct receptors for high-affinity agonist and antagonist binding.     

Comparison of the Binding of [3H]Spiperone and [3H]Domperidone in Homogenates of Mammalian Retina and Caudate Nucleus

Abstract— The specific binding of [³H]spiperone and [³H]domperidone, as defined by 1 μ m -(+)butaclamol, was compared in homogenates of bovine retina and caudate nucleus. Scatchard analyses of saturation data for [³H]spiperone binding yielded dissociation constants ( K _d) of 0.35 n m in the retina and 0.64 n m in the caudate nucleus. Comparison of the maximum number of binding sites (B_max) present in each tissue indicated that the density of sites in bovine caudate nucleus (270 fmol/mg protein) was approximately three times higher than in bovine retina (92 fmol/mg protein). This difference was even more marked in guinea pig tissues, with a ratio of 7:1 between corpus striatum and retina. The pharmacological analysis of [³H]spiperone binding in both the bovine retina and caudate nucleus indicated an interaction with dopaminergic rather than serotonergic sites. However, inhibition curves obtained to dopaminergic agonists in the bovine retina were significantly steeper than those observed in the bovine caudate nucleus, as reflected in the greater Hill coefficients obtained for these agents in the retina. Furthermore, only a small amount of specific [³H]domperidone binding was observed in either the bovine caudate nucleus or the guinea pig striatum, whilst no specific [³H]domperidone binding was detectable in homogenates of either bovine or guinea pig retina. These data suggest that the retina possesses only a small population of dopaminergic D2 sites and that these binding sites may differ from those present in the caudate nucleus.     

Imipramine-Induced Changes in 5-HT2 Receptor Sites and Inositoltrisphosphate Levels in Rat Brain

The effect of chronic administration of Imipramine on [³H]Spiperone binding to 5-HT₂ sites and inositoltrisphosphate (IP₃) levels in rat cerebral cortex was studied. Our data shows that treatment with imipramine (5 mg/kg body weight, intraperitoneally) for 30 days significantly down regulates 5-HT₂ receptors sites (262 ± 29 fmol/mg protein) in cerebral cortex (38%), compared to control rats (425 ± 60 fmol/mg protein., P < 0.001). However there was no significant change in the affinity of [³H]-Spiperone binding (kd) to 5-HT₂ sites in cerebral cortex after exposure to imipramine (Kd = 0.84 ± 0.11 nM). It is also observed that imipramine treatment significantly reduces 5-HT stimulated [³H]IP₃ formation in cerebral cortex (6,411 ± 708 dpm/mg protein), compared to the saline treated rats (12,238 ± 1,544 dpm/mg protein; P < 0.001), with concomitant decrease in Pdtlns-4–5-P₂. This study suggests that the therapeutic action of imipramine in brain might be by reducing hypersensitivity of 5-HT₂ receptors by down regulation, which leads to reduced levels of inositolphospholipids. This inturn reduces the levels of IP₃. In conclusion, imipramine acts at presynaptic site by blocking the reuptake of serotonin and at post synaptic site it downregulates 5-HT₂ sites with decreased IP₃ levels after chronic exposure.     

[3H]Fluphenazine Binding to Brain Membranes: Simultaneous Measurement of D-1 and D-2 Receptor Sites

[3H]Fluphenazine was used to label both D-1 and D-2 dopamine receptors in mouse striatal membranes. The D-1 and D-2 specific binding of [3H]fluphenazine was discriminated by the dopamine antagonists SCH-23390 (D-1 selective) and spiperone (D-2 selective). Saturation analyses of these two sites yielded a D-1 receptor density in mouse striatum of 1,400 fmol/mg of protein and a D-2 receptor density of 700 fmol/mg of protein. The affinity of [3H]fluphenazine for the D-2 site was slightly greater than for the D-1 site; the equilibrium dissociation constant (KD) was 0.7 versus 3.2 nM, respectively. Assay conditions are described that reduce nonspecific binding of [3H]fluphenazine to acceptable levels (35% of total binding at 1 nM [3H]fluphenazine). By comparison of displacement curves from a series of dopaminergic and nondopaminergic ligands, the pharmacological specificity of [3H]fluphenazine binding in mouse striatum was demonstrated to be dopaminergic. Only small amounts of dopamine-specific (apomorphine-sensitive) [3H]fluphenazine binding were found in other brain regions. However, chlorpromazine displaced considerable [3H]fluphenazine from all brain regions, including cerebellum, suggesting the presence of a [3H]fluphenazine binding site with a phenothiazine specificity.     

Effects of Chronic Striatal Kainate Lesions on Some Dopaminergic Parameters and Enkephalin Immunoreactive Neurons in the Basal Ganglia

Abstract: The chronic effects of kainate-induced lesions of the neostriatum have been evaluated in rats 12 months following the injection of kainic acid. Light microscopical analysis revealed marked disappearance of nerve cells in the neostriatum, with some cells remaining within the medial and lateral zone of the neostriatum and in the most ventral part. The rest of the markedly atrophied neostriatum was mainly made up of densely packed myelinated nerve bundles. Tyrosine hydroxylase immunoreactivity was used as a marker for dopamine neurons and revealed that tyrosine hydroxylase immunoreactive nerve terminals remained between the axon bundles in the striatum and that tyrosine hydroxylase immunoreactive nerve cell bodies in the substantia nigra seemed intact. Studies on enkephalin immunoreactive neurons revealed a marked disappearance of such nerve cells and nerve terminals within the neostriatum. Neurochemical analysis showed a clearcut reduction in the number of dopamine receptors as evaluated by studies on both [³H]spiperone binding and on [³H]ADTN binding. Dopamine levels remained unchanged while choline acetyltransferase activity was reduced significantly. Taken together, the present findings demonstrate that the chronically kainate lesioned striatum is characterized by a substantial loss of enkephalin immunoreactive and cholinergic nerve cells and a marked reduction in the number of dopamine receptors. These findings are discussed in relation to neurochemical and therapeutic aspects of Huntington's disease.     

Short Communication Occupation of Dopamine Receptors by N-n-Propylnorapomorphine or Spiperone and Acetylcholine Levels in the Rat Striatum

In an attempt to quantify the interactions between dopaminergic and cholinergic processes, the consequences of complete or partial activation (with N-n-propylnorapomorphine) or blockade (with spiperone) of dopamine receptors for the acetylcholine levels in the rat striatum were studied. The number of specific striatal binding sites (receptors) of spiperone was nearly three times that of N-n-propylnorapomorphine (76 and 26 pmol g-1 wet weight, respectively). The agonist produced a significant increase in the striatal levels of acetylcholine, but there was no simple relationship between receptor binding and these levels. A linear negative correlation was found between the striatal levels of acetylcholine and specific spiperone binding, showing that further receptor blockade induces a decrease in acetylcholine levels, which is independent of the receptors already occupied by the antagonist. The results of this study are evidence that one striatal dopamine receptor regulates the metabolism of at least 400 molecules of acetylcholine.