Comparison of Three 18F-Labeled Butyrophenone Neuroleptic Drugs in the Baboon Using Positron Emission Tomography

The butyrophenone neuroleptics spiroperidol, benperidol, and haloperidol were radiolabeled with fluorine-18 and studied in baboon brain using positron emission transaxial tomography (PETT). Pretreatment of the baboon with a high pharmacological dose of (+)-butaclamol reduced the specifically bound component of radioactivity distribution in the striatum to approximately the radioactivity distribution found in the cerebellum. Comparative studies of brain distribution kinetics over a 4-h period indicated that either [18F]spiroperidol or [18F]benperidol may be suitable for specific labeling of neuroleptic receptors. In an 8-h study with [18F]spiroperidol, striatal radioactivity did not decline, suggesting that spiroperidol either has a very slow dissociation rate or that it binds irreversibly to these receptors in vivo. [18F]Haloperidol may not be suitable for in vivo PETT studies, because of a relatively high component of nonspecific distribution and a faster dissociation from the receptor. Analysis of 18F in plasma after injection of [18F]spiroperidol indicated rapid metabolism to polar and acidic metabolites, with only 40% of the total radioactivity being present as unchanged drug after 30 min. Analysis of the metabolic stability of the radioactively labeled compound in rat striatum indicated that greater than 95% of [18F]spiroperidol remains unchanged after 4 h.     

Schistosoma mansoni: differences in acetylcholine, dopamine, and serotonin control of circular and longitudinal parasite muscles

The physiological and pharmacological properties of circular and longitudinal somatic musculature in adult male Schistosoma mansoni were compared using cut muscle sections. Carbachol reduced tone in both circular and longitudinal muscle, but was without effect on circular muscle bathed in high Mg2+, indicating that cholinergic receptors were not associated with circular muscle membrane. 5-Hydroxytryptamine (5-HT) induced rhythmic contractile activity in both sets of muscle. It decreased muscle tone in circular muscle but increased the tone of longitudinal muscle. Metergoline blocked 5-HT effects on both sets of muscle. 5-HT continued to be effective on both sets of muscle bathed in high-Mg2+ medium, indicating that serotonergic receptors were present on both circular and longitudinal muscle membranes. Dopamine decreased both circular and longitudinal muscle tone. Its effects on circular muscle were still present after exposure to high Mg2+, but its effects on the longitudinal muscle were significantly reduced, leading to the conclusion that dopaminergic sites were probably associated with circular muscle membrane but not that of longitudinal muscle. Also, spiroperidol blocked stimulus responsiveness of the circular muscle but not that of the longitudinal muscle. From these studies it appears that there are significant physiological and pharmacological differences between circular and longitudinal muscles in the adult male schistosome.     

Ascorbate-Induced Lipid Peroxidation and Inhibition of [3H]Spiroperidol Binding in Neostriatal Membrane Preparations

Sodium ascorbate caused an increased lipid peroxidation and a large decrement in [3H]spiroperidol binding in a rat neostriatal membrane preparation (preparation C). Both effects were greater at intermediate (0.05 and 0.5 mM) than at higher or lower ascorbate concentrations. In contrast, in another neostriatal membrane preparation (preparation A), there was no loss of [3H]spiroperidol binding and only a small increase in lipid peroxidation caused by ascorbate. However, both the ascorbate-induced increase in lipid peroxidation and loss of [3H]spiroperidol binding were greatly enhanced in preparation A by the addition of iron salts. In experiments designed to explore reasons for these apparent discrepancies, we discovered that the method of tissue preparation was a critical factor. The ascorbate effects were consistently greater in a tissue preparation which was originally homogenized in an isotonic sucrose medium and centrifuged, and the cell debris discarded (as was done in preparation C), than in one in which the tissue was homogenized in a hypotonic medium and in which no low-speed centrifugation was done (as was done in preparation A). In other experiments, of several cations tested, only ferrous and ferric potentiated the above-described effects of ascorbate. Some ascorbic acid derivatives (e.g., isoascorbic acid) had properties similar to those of ascorbic acid, whereas several reducing agents could, in the presence of added iron salts, cause both a lipid peroxidation and a loss of [3H]spiroperidol binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Soluble Spiroperidol Binding Factors from Bovine Caudate Nucleus

Several properties of soluble spiroperidol binding factors separated from bovine caudate nucleus have been investigated by a previously unreported procedure. Data consistent with high particle weight and rapid binding equilibration are reported for high-affinity (+)butaclamol-sensitive components of a digitonin extract. A slower sedimenting component is found that also exhibits high affinity for spiroperidol but is not sensitive to (+)butaclamol. Centrifugation of a caudate nucleus homogenate yields a supernatant that appears to contain a component that exhibits spiroperidol binding that is more sensitive to displacement by (-) than by (+)butaclamol. The procedure used effects rapid separation of bound from unbound tritiated ligand on short columns of Sephadex G-15 followed by extrusion and sectioning of the Sephadex. The radioactivity remaining with each section is determined. The procedure is very rapid; the addition of active phases or the changing of the ionic environment, which may disturb the equilibrium, is avoided; and recovery of the protein free of bound ligand is easily affected.     

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D₁ and D₂ receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D₁ receptor antagonist, [³H]SCH 23390, and a dopamine D₂ receptor antagonist, [³H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K _D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [³H]SCH 23390 and [³H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D₁ or D₂ receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [³²P]NAD demonstrated predominant labeling of bands of M_r 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of G_s and G_i proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D₂ receptor agonists, was present in the CVs. These findings suggest that the dopamine D₁ and D₂ receptors in the CVs couple with adenylate cyclase via G_s/G_i protein.     

Serotonin-Elicited Amplification of Adenylate Cyclase Activity in Hippocampal Membranes from Adult Rat

The activity of the adenylate cyclase located in membranes prepared from hippocampus of adult rat can be stimulated by serotonin (5-HT) (Ka = 4 X 10(-7) M). The maximal effect is obtained with 10 microM 5-HT. Freezing of the tissue decreases the 5-HT stimulation; this stimulation is optimal in the presence of 82.5 mM Tris-maleate buffer (pH 7.4) and 50 microM GTP. The adenylate cyclase activity of membranes prepared from cortex, hypothalamus, and colliculi of adult rats is not significantly stimulated by 5-HT. Dopamine (DA) also stimulates adenylate cyclase located in hippocampal membranes; its effect can be blocked by haloperidol (10(-6) M), which fails to inhibit 5-HT stimulation. Moreover, p-chlorophenylalanine treatment for 2 weeks or selective lesion of 5-HT axons afferent to the hippocampus increases the Vmax of 5-HT stimulation, but fails to change that of DA stimulation. The 5-HT stimulation can be inhibited by metergoline, spiroperidol, and pizotyline (10(-6) M), but not by the same concentrations of mianserin, ketanserine, alprenolol, phenoxybenzamine, and mepyramine. The 5-HT stimulation of adenylate cyclase of hippocampal membranes can be mimicked by tryptamine, 5-methoxytryptamine, bufotenine, and to a lesser extent by LSD; N-methyltryptamine, N-methyltryptophan, and 5-hydroxytryptophan are inactive. Studies with kainic acid suggest that the 5-HT recognition site (5-HT1) linked to adenylate cyclase is located on the membrane of intrinsic hippocampal neurons.     

Serotoninergic System in Scrapie-Infected Hamsters

Hamsters inoculated with scrapie virus show a dramatic hypersensitivity to serotoninergic drugs, developing a behavioral syndrome not unlike that obtained with pharmacologically induced lesions of the raphe nuclei. In an attempt to explain the state of hypersensitivity and to determine whether or not serotoninergic neurons were targets of the scrapie virus, pre- and postsynaptic serotoninergic sites were studied in the cerebral cortices of scrapie-infected and sham-inoculated hamsters. [3H]Imipramine binding and the uptake of endogenous 5-hydroxytryptamine (5-HT, serotonin) in synaptosomes prepared from scrapie-inoculated animals were not different from those of controls. This suggests integrity of the serotoninergic neurons in scrapie-infected hamsters. In contrast, affinity for the 5-HT1 receptor (which modulates inhibitory response) was diminished whereas that for the 5-HT2 receptor (which modulates excitatory response) was increased. This "imbalance" between the two receptors which is amplified in in vivo responses may account for the 5-HT hypersensitivity. The alteration in the affinity of the two postsynaptic 5-HT receptors supports the observation that scrapie virus alters cell plasma membranes.     

Monoclonal Antibodies with High Affinity for Spiroperidol

A diverse panel of monoclonal antibodies was obtained from BALB/c mice immunized with two haptens structurally related to spiroperidol (SPD). Bromoacetyl derivatives of aminospiroperidol (NH2SPD) and N-amino-phenethylspiroperidol (NAPS) were synthesized to couple the haptens covalently to a protein carrier for immunization, thereby maintaining the butyrophenone portion of the immunogen. Hybridomas were selected based on their ability to secrete antibody that binds [3H]SPD with high affinity. Equilibrium dissociation constants for these antibodies ranged from 0.2 to greater than 100 nM. The antigen binding sites of the anti-NH2SPD and anti-NAPS antibodies were characterized in studies of the inhibition of the binding of [3H]-SPD by a series of ligands that are either (a) structurally related to SPD or (b) structurally unrelated to the butyrophenones but known to be selective antagonists of the D2 subtype of dopamine receptor. Based on the patterns of inhibition of the binding of [3H]SPD by these compounds, 12 classes of antibody combining sites were identified. Most of these antibodies bound butyrophenones with high affinity. One anti-NH2SPD and four anti-NAPS antibodies also bound domperidone, a nonbutyrophenone that has a high affinity for D2 receptors. None of the antibodies bound clebopride or sulpiride, D2-selective antagonists of the benzamide class, or the agonist dopamine.     

Specific Binding of [11C]Spiroperidol in Rat Brain In Vivo

Spiroperidol labeled with carbon-11, a short-lived positron-emitting radionuclide, was used to determine the time course of specific binding of this radioligand to the neuroleptic receptor in vivo in the rat. The three major bran pools--specifically bound, nonspecifically bound, and free (unbound)--were determined over a 60-mm time course by a rapid filtration technique, utilizing (-)- and (+)-butaclamol pretreatments to assess total and nonspecifically bound activities, respectively, in striatum and cerebellum. The ratio of specifically to nonspecifically bound pools in the striatum was 4.1 at 30 min and 5.1 at 60 min. Thus [11C]spiroperidol may be useful for labeling neuroleptic receptors in vivo for serial studies using positron emission transaxial tomography.     

Inhibition of [3H]Spiroperidol Binding by In Vitro Addition of Ethanol

: The in vitro addition of ethanol at concentrations greater than 0.2% produced an inhibition of [³H]spiroperidol binding. This inhibition was competitive, lowering the affinity without altering the maximum number of sites. Both the rates of association and dissociation were altered such that the ligand spent less time attached to the receptor. Since the binding was competitive, little inhibition was seen at ligand concentrations greater than 300 pm . These results are important for in vitro studies when drugs are dissolved in ethanol, and also for in vivo studies on the effects of ethanol.