High-Affinity Choline Transport Sites: Use of [3H]Hemicholinium-3 as a Quantitative Marker

Abstract: High-affinity choline transport (HAChT), the rate-limiting and regulatory step in acetylcholine (ACh) synthesis, is selectively localized to cholinergic neurons. Hemicholinium-3 (HC3), a potent and selective inhibitor of HAChT, has been used as a specific radioligand to quantify HAChT sites in membrane binding and autoradiographic studies. Because both HAChT velocity and [³H]HC3 binding change as in vivo activity of cholinergic neurons is altered, these markers are also useful measures of cholinergic neuronal activity. Evidence that [³H]HC3 is a specific ligand for HAChT sites on cholinergic terminals is reviewed. The ion requirements of HAChT and [³H]HC3 binding indicate that sodium and chloride are required for recognition of both choline and [³H]HC3. A common recognition site is also indicated by the close correspondence of the potency of HC3 and choline analogues for inhibiting both HAChT and [³H]HC3 binding. The parallel regional distributions of both markers in adult brain, during development and after specific lesions, all indicate specific cholinergic localization. The close association of HAChT and [³H]HC3 binding sites is also supported by parallel regulatory changes occurring after in vivo drug treatments and in vitro depolarization. Overall, the data indicate a close association between HAChT and [³H]HC3 binding and are consistent with the sites being identical. Methodologic considerations in using [³H]HC³ as a ligand and considerations in interpretation of results are also discussed.     

Identification of Sodium-Dependent, High-Affinity Choline Uptake Sites in Rat Brain with [3H]Hemicholinium-3

[3H]Hemicholinium-3 (HC-3) was used to label sodium-dependent, high-affinity choline uptake sites in regions of rat brain. Autoradiography revealed a high density of [3H]HC-3 binding sites in brain regions with a high density of cholinergic terminals, such as the interpeduncular nucleus, caudate-putamen, and olfactory tubercle. This distribution of [3H]HC-3 binding sites was in close agreement with the amounts of choline acetyltransferase in specific nuclei and subregions of rat brain. Destruction of presynaptic cholinergic projections in the cerebral cortex and the basal ganglia by injection of excitotoxins reduced [3H]HC-3 binding by 40-50%. These data indicate that sodium-dependent [3H]HC-3 binding sites are related to the choline transport system present in cholinergic neurons.     

Regulation of Rat Brain Synaptosomal [3H]Hemicholinium-3 Binding and [3H]Choline Transport Sites Following Exposure to Choline Mustard Aziridinium Ion

Abstract: Choline uptake by cholinergic nerve terminals is increased by depolarization; the literature suggests that this results from either the appearance of occult transporters or the increased activity of existing ones. The present experiments attempt to clarify the mechanism by which choline transport is regulated by testing if the preexposure of synaptosomes to choline mustard aziridinium ion prevents the stimulation-induced appearance of hemicholinium-3 binding sites and/or choline transport activity. Choline mustard inhibited irreversibly most of the “ground-state” (basal) high-affinity choline transport but only 50% of “ground-state” hemicholinium-3 binding sites. Exposure of both striatal and hippocampal synaptosomes to the mustard, before stimulation, inhibited K⁺-stimulated increases in choline transport and of [³H]hemicholinium-3 binding. We conclude that the mechanism by which choline transport is regulated involves the increased activity of a pool of transport sites that are occluded to hemicholinium-3 but are available to choline mustard aziridinium ion, and presumably to choline, before stimulation. However, the concentration of mustard needed to inhibit the stimulation-induced increase of [³H]hemicholinium-3 binding and choline transport was lower for striatal synaptosomes than for hippocampal synaptosomes. In the absence of extracellular Ca²⁺ or presence of high Mg²⁺ levels, the choline mustard did not prevent the appearance of extra striatal hemicholinium-3 binding sites. Also, high Mg²⁺ levels removed the ability of the mustard to inhibit K⁺-stimulated increases of either [³H]hemicholinium-3 binding or choline transport by hippocampal synaptosomes. In contrast, the preexposure of hippocampal synaptosomes to the mustard in the presence of a calcium ionophore (A23187) reduced the concentration of inhibitor needed to prevent the activation of [³H]hemicholinium-3 binding and choline uptake. Thus, we conclude that the ability of the choline mustard to alkylate the pool of choline transporters that are activated by stimulation appears dependent on the entry of extracellular Ca²⁺.     

Affinity Labelling and Identification of the High-Affinity Choline Carrier from Synaptic Membranes of Torpedo Electromotor Nerve Terminals with [3H]Choline Mustard

The physiological mechanisms regulating activity of the sodium-dependent, high-affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion [( 3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium-sensitive, and sodium-, time-, and temperature-dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30 degrees C. Separation of the 3H-labelled membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of approximately 42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC-3. Two other 3H-labelled polypeptides were detected, with apparent molecular masses of approximately 58,000 and 90,000 daltons; radiolabelling of the former was also HC-3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.     

[3H]Choline Uptake and Metabolism in Nonsynaptic Regions of a Crustacean Sensory Nerve

Abstract: The posterior stomach nerve (PSN) is a crustacean sensory nerve containing about 60 cholinergic neurons, which are devoid of synaptic interactions. Kinetic analysis shows that the PSN takes up [³H]choline by both low-affinity ( K _m= 163 μM) and high-affinity (Na⁺-dependent) ( K _m= 1 μM) processes. The capacity of the high-affinity system is only about 1% that of the low-affinity system. The high-affinity system is not tightly coupled to acetylcholine (ACh) synthesis, and it appears that both ACh and phosphorylcholine are formed from an intracellular pool of choline, which is fed by both uptake systems. There are differences in the rates of [³H]choline uptake and ³H metabolite accumulation between regions of the PSN that contain neuronal cell bodies and those that do not. These differences may arise from differences in the relative proportion of neuronal to nonneuronal tissue in each nerve region.     

Solubilization and Characterization of a [3H]Hemicholinium-3 Binding Site in Rat Brain

A sodium-dependent high-affinity [3H]-hemicholinium-3 ([3H]HCh-3) binding site was solubilized from rat striatal synaptic plasma membranes by 0.2% deoxycholate. Deoxycholate solubilization of the [3H]HCh-3 binding site was dependent upon both detergent concentration and ionic strength of the solubilization medium. Specific [3H]HCh-3 binding to the solubilized preparation was both sodium- and chloride-dependent and saturable, exhibiting an affinity of 14.2 nM and a capacity (Bmax) of 695 fmol/mg protein. Choline and other analogs inhibited specific [3H]HCh-3 binding to the solubilized preparation in a concentration-dependent manner with the similar rank order of potency observed in crude synaptic membranes. Treatments known to disrupt both protein and lipid moieties resulted in diminished specific [3H]HCh-3 binding. These results suggest that the characteristics of the solubilized [3H]HCh-3 binding site are similar to those of the membrane-bound site.     

Binding of [3H]Hemicholinium-3 to the High-Affinity Choline Transporter in Electric Organ Synaptosomal Membranes

Sodium-dependent binding of [3H]hemicholinium-3 was observed to be 10-fold higher with presynaptic membranes from the electric organ than with electroplaque membranes and this binding site copurified with synaptosomal membranes. The KD for specific [3H]hemicholinium-3 binding was found to be 31 +/- 4 nM and the Bmax, 5.0 +/- 0.2 pmol/mg protein; a Ki of 16 nM was estimated for hemicholinium-3 as a competitive inhibitor of high-affinity choline transport in electric organ synaptosomes. Choline and choline analogues were equally potent as inhibitors of [3H]choline uptake and [3H]hemicholinium-3 binding. Tubocurarine and oxotremorine also inhibited uptake and binding, but carbachol was without effect in both tests. These findings suggest that [3H]hemicholinium binds to the high-affinity choline transporter present at the cholinergic nerve terminal membrane. A comparison of maximal velocities for choline transport and the maximal number of hemicholinium-3 binding sites indicated that the high-affinity choline transporter has an apparent turnover number of about 3s-1 at 20 degrees C under resting conditions. The high transport rates observed in electric organ synaptosomes are likely due to the high density of high-affinity choline transporters in this tissue, estimated on the basis of [3H]hemicholinium-3 binding to be of the order of 100/micron2 of synaptosomal membrane.     

Stereoselectivity of the Inhibition of [3H]Hemicholinium-3 Binding to the Sodium-Dependent High-Affinity Choline Transporter by the Enantiomers of a- and β-Methylcholine

Abstract: In a previous report, we showed that the enantiomers of α- and β-methylcholine inhibited choline uptake with Stereoselectivity, but that their transport by the choline carrier of nerve terminals showed stereospecificity. The present experiments used the same choline analogues to determine if either of the above characteristics pertains to their ability to interact with the [³H]-hemicholinium-3 binding site present on striatal membranes and synaptosomes. [³H]Hemicholinium-3 binding to striatal membranes could be inhibited stereoselectively by the enantiomers of β-methylcholine, but R (+)-α-methyl-choline was little better than its enantiomer in this test. However, [³H]hemicholinium-3 binding to striatal synaptosomes was inhibited stereoselectively by the enantiomers of both α- and β-methylcholine. This difference between the properties of [³H]hemicholinium-3 binding to membranes or to synaptosomes appears related to the presence of two ligand binding states. The [³H]hemicholinium-3 binding site could be shifted to a low-affinity state by ATP treatment and to a high-affinity state by EDTA washing. When the [³H]hemicholinium-3 binding site existed in its low-affinity state, binding was inhibited stereoselectively by the enantiomers of both a- and β-methylcholine, but when shifted to its high-affinity state, it was inhibited stereoselectively only by the enantiomers of β–methylcholine. We conclude that hemicholinium-3 interacts with the substrate recognition site of the high-affinity choline transporter, but that the Stereoselectivity of this site changes depending on its affinity state.     

Characteristics of [3H]Hemicholinium-3 Binding to Rat Striatal Membranes: Evidence for Negative Cooperative Site-Site Interactions

The characteristics of [3H]hemicholinium-3 ([3H]HC-3) interactions with rat striatal membranes were investigated. Under the described assay conditions, [3H]-HC-3 binds with a saturable population of membrane binding sites having the following regional distribution: striatum much greater than hippocampus greater than or equal to cerebral cortex greater than cerebellum. The specific binding of [3H]HC-3 showed an obligatory requirement for NaCl; other halide salts of sodium or KCl failed to substitute for NaCl. The Scatchard transformation of saturation isotherm data generated a curvilinear plot with high- and low-affinity components of binding. The dissociation of [3H]HC-3 at infinite dilution was also multiexponential. The dissociation could, however, be accelerated if unlabeled HC-3 was included in the diluting buffer, and this increase in dissociation appeared to be dependent on the concentrations of unlabeled HC-3 used, with the maximal increase demonstrable at 100 nM. The dissociation was also dependent on the fractional saturation of binding sites with labeled HC-3, such that, at higher fractional saturation of binding sites, the overall dissociation was faster and the difference in the dissociation observed between "dilution only" and "dilution + unlabeled HC-3" was reduced. This occupancy-dependent change in dissociation could also be influenced by temperature and pH. Based on the results of these kinetic studies, the steady-state [3H]HC-3 binding data were analyzed for a homogeneous population of binding sites undergoing site-site interactions of the negative cooperative type. Such an analysis yielded a KD of 9.3 nM for the high-affinity state and a KD of 22.8 nM for the low-affinity state of binding sites, with a Bmax of 434 fmol/mg of protein. Competitive binding studies showed that unlabeled HC-3 was most potent in displacing [3H]HC-3, followed by choline. Other drugs known to have little influence on the synaptosomal sodium-dependent high-affinity choline uptake system (SDHACU) had no significant effect on [3H]HC-3 binding sites. Similarities in ionic dependencies, regional distributions, and pharmacological selectivities of [3H]HC-3 binding with synaptosomal SDHACU suggest that [3H]HC-3 selectively labels SDHACU sites located on presynaptic cholinergic neurons in rat CNS. We suggest that the two affinity states of [3H]HC-3 binding sites represent the different "functional" states of the SDHACU system. The binding of HC-3 (or choline) with the high-affinity state of the binding sites induces negative cooperative site-site interactions among the binding sites, resulting in the formation of a low-affinity binding state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of [3H]Choline and d-[3H]Aspartate Efflux from Guinea Pig and Human Neocortex

The outflow of [3H]choline ([3H]Ch) evoked by electrical field stimulation and the efflux of D-[3H]Asp induced by 35 mM KCl and 1-10 microM ouabain were studied in human and guinea pig cortical slices, kept under identical experimental conditions. [3H]Ch outflow was significantly lower whereas D-[3H]Asp efflux was significantly higher in humans than in guinea pigs. This suggests a different proportion of the two neuronal systems in these two species. Blockade of muscarinic autoreceptors with atropine increased, whereas stimulation of alpha 2 receptors with norepinephrine (NE) reduced, the evoked [3H]Ch outflow to the same extent in human and guinea pig cortical slices. Conversely, NE did not affect ouabain-induced D-[3H]Asp efflux, suggesting that an alpha 2-mediated control is not operative in the glutamatergic cortical structures. Desmethylimipramine, 2-5 microM, was able to increase [3H]Ch outflow through atropine-like mechanisms only in the human. This drug at 20-50 microM inhibited [3H]Ch and D-[3H]Asp efflux in both species, through mechanisms unrelated to its monoamine reuptake blocking properties. Thus, similarities and differences can be detected between humans and guinea pigs with regard to (a) the relative potency of the cholinergic and acidic amino acidergic signals and (b) the modulation of neurotransmitter outflow by drugs acting on auto- and the heteroreceptors.     

Distribution of Specific High-Affinity Binding Sites for [3H]Imipramine in Human Brain

Abstract: [³H]Imipramine binds with high affinity to membranes from different regions of the human brain. The highest density of binding sites was observed in the hypothalamus and substantia nigra and the lowest density in the white matter and cerebellum. As found in rat brain, tricyclic antidepressant drugs are potent inhibitors of [³H]imipramine binding. Atypical antidepressants are, however, much weaker at inhibiting the specific binding. The [³H]imipramine binding site in human cortex is apparently identical to the site already described in the rat brain and in human platelets.     

High-Affinity Choline Transport Regulation by Drug Administration During Postnatal Development

High-affinity choline transport sites specifically bind [3H]hemicholinium-3. Hemicholinium-3 binding sites are regulated by in vivo drug treatments in the same manner as these drugs alter acetylcholine release and high-affinity choline transport. The current study examines regulation of binding sites by in vivo drug administration for adult, day 15, and day 5 rats. Drugs or saline were administered intraperitoneally, and striatal and cortical membrane preparations were assayed. Control [3H]hemicholinium-3 binding increases twofold between postnatal days 5 and 15 only in striatum. After day 15, binding increases 2.7-fold in cortex and striatum. Nicotine treatment increases striatal and cortical hemicholinium-3 binding at all three ages, with greater percent increases at day 5. Haloperidol increases binding only in striatum, again with larger effects at day 5. Both striatal and cortical binding are reduced by oxotremorine; however, the magnitude of this effect is unchanged during development. Pentobarbital reduces binding only in striatum, with no developmental change. Atropine and apomorphine do not change binding from control values. In summary, all drug treatments effective in adults were already effective by day 5. Cholinergic terminals present early in development are regulated by similar nicotinic and muscarinic cholinergic, dopaminergic, and sedative-hypnotic mechanisms as the adult. Changes in magnitude may be due to changes in drug metabolism or to developmental differences in regulation.     

Characterization of "High-Affinity"[3H]Ouabain Binding in the Rat Central Nervous System

The characteristics of [3H]ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of "high-affinity" binding sites with an apparent binding affinity (KD) of 10.4 +/- 0.9 nM and an estimated binding capacity (Bmax) of 7.6 +/- 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. [3H]Ouabain binding to rat brain was sodium- and ATP-dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific [3H]ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K-ATPase. To assess whether the high-affinity binding sites for [3H]ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal [3H]ouabain binding was examined. Kainic acid lesions of the striatum reduced [3H]ouabain binding to striatal homogenates by 79.6 +/- 1.6%. This suggests that the "high-affinity" [3H]ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high-affinity binding of [3H]ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K-ATPase.     

Lack of Selectivity Between the Uptake of [3H] Adrenaline and [3H]Noradrenaline into Rat Hypothalamic Slices

The uptake of [3H]adrenaline and [3H]noradrenaline into rat hypothalamic slices was compared for determination of whether adrenaline uptake was independent of uptake into noradrenergic neurones. Kinetic analysis revealed a similar high-affinity uptake process for both adrenaline and noradrenaline, with Km and Vmax values within similar ranges. These uptakes were inhibited by desipramine and maprotiline in a dose-dependent manner, but the selective dopamine and 5-hydroxytryptamine uptake inhibitors benztropine and fluoxetine, respectively, were without effect. Competition for uptake sites by unlabelled adrenaline with [3H]adrenaline and [3H]-noradrenaline and by unlabelled noradrenaline with [3H]-adrenaline and [3H]noradrenaline was very similar. Lesioning of the major adrenaline-containing cell group (C1 cell group) decreased the hypothalamic adrenaline concentration but had no effect on hypothalamic [3H]adrenaline or [3H]noradrenaline uptake. The results suggest that exogenous adrenaline is largely taken up by high-affinity sites on noradrenergic nerve terminals.     

Interleukin-2 Modulates Evoked Release of [3H]Dopamine in Rat Cultured Mesencephalic Cells

Abstract: Mesencephalic cell cultures were used as a model to investigate the effects of interleukin-2 (IL-2) on evoked release of [³H]dopamine ([³H]DA) and γ-[³H]-aminobutyric acid ([³H]GABA). At low concentrations (10^?13-10^?12M), IL-2 potentiated [³H]DA release evoked by the excitatory amino acids N-methyl-D-aspartate (NMDA) and kainate, whereas higher IL-2 concentrations (10^?9-10^?8M) had no effect. IL-2 (10^?14-10^?8M) modulated K⁺-evoked [³H]DA release in a biphasic manner, with low concentrations (10^?12-10^?11M) of IL-2 potentiating and higher concentrations (10^?9-10^?8M) inhibiting K⁺-induced [³H]DA release. IL-2 (10^?14-10^?8M) by itself failed to alter spontaneous [³H]DA release. The inhibition by IL-2 of K⁺-evoked [³H]DA release was reversible and not due to neurotoxicity, as preexposure to IL-2 (10^?8M) had no significant effect on the subsequent ability of dopaminergic cells to take up and to release [³H]DA. Under our experimental conditions, IL-2 (10^?8 M) did not alter Ca²⁺-independent [³H]GABA release evoked by either K⁺ or NMDA. The results of this study indicate that IL-2 is able to potentiate [³H]DA release evoked by a number of different stimuli, including K⁺ depolarization and activation of both NMDA and non-NMDA receptor subtypes in mesencephalic cell cultures. IL-2 is active at very low concentrations, a finding that indicates a potent effect of IL-2 on dopaminergic neurons and implicates a physiological role for this cytokine in the modulation of DA release.     

Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nicotine Indirectly Inhibits [3H]Dopamine Uptake at Concentrations That Do Not Directly Promote [3H]Dopamine Release in Rat Striatum

The effects of both (-)- and (+)-nicotine isomers were examined on in vitro uptake and release of [3H]dopamine in rat striatum. Both isomers inhibited uptake of [3H]dopamine in chopped tissue at concentrations well below those necessary for promoting release of preloaded [3H]dopamine. (-)-Nicotine was more potent than (+)-nicotine both at inhibiting uptake and at promoting release. Unlike other dopamine uptake inhibitors, however, nicotine inhibited only 50% of the total uptake. In the presence of 1 nM nicotine, the residual [3H]dopamine uptake was less sensitive to inhibition by cocaine than uptake in the absence of nicotine. Nicotine did not compete against the binding of [3H]GBR 12935, a selective dopamine uptake inhibitor. The nicotinic receptor agonists carbachol and 1,1-dimethyl-4-phenylpiperazinium iodide also inhibited uptake, whereas the nicotinic antagonists chlorisondamine and mecamylamine blocked nicotine's effect. Thus, the effect of nicotine on dopamine uptake appears to be mediated by a receptor similar to the nicotinic acetylcholine receptor. These receptors do not seem to be on the terminals that are accumulating dopamine, however, since tetrodotoxin prevented the effect of nicotine on [3H]dopamine uptake and nicotine had no effect on uptake in a synaptosomal preparation.     

Ionic and Metabolic Requirements for High-Affinity Choline Uptake and Acetylcholine Synthesis in Nerve Terminals at a Neuromuscular Junction

Abstract: We have shown previously that in the chick ciliary nerve-iris muscle preparation Na⁺-dependent high-affinity choline uptake was confined to the nerve terminals. In this paper the sodium-dependent high-affinity choline uptake (SDHACU), which is coupled to acetylcholine (ACh) synthesis, was further characterized by measuring uptake of [³H]choline and its conversion to [³hjach under a variety of ionic and metabolic perturbations. Mannitol equilibration with the extracellular space was found to occur in less than 1 min in this preparation. Na⁺-dependent choline (Ch⁺) uptake was shown to be linear for 16 min and to reach an equilibrium before Na⁺-independent Ch⁺ uptake, which continued to increase for 60 min. Elevated [K⁺]₀ concentrations inhibited Ch⁺ uptake and ACh synthesis. Glycolytic and respiratory inhibitors also reduced both processes, as did ouabain and omission of [K⁺]₀. Incubation conditions that reduce transmitter release had no effect on inhibition by high [K⁺]₀. Reduction of SDHACU and sodium-dependent ACh synthesis by depolarization with high [K⁺]₀ or by inhibition of Na, K-ATPase implies that the electrochemical gradients for Ch⁺ and Na⁺ are important in providing a driving force for high-affinity Ch⁺ uptake. The inhibition by metabolic blockers suggests active transport, but the effects may be indirect, caused by reduced Na, K-ATPase activity and alterations in membrane potential. While most metabolic inhibitors exerted parallel effects on both Ch⁺ uptake and ACh synthesis, in some cases Ch⁺ uptake was more strongly inhibited than ACh synthesis. This occurred in preparations incubated with high [K⁺]₀ and ouabain. Na⁺-dependent Ch⁺ uptake and ACh synthesis were found to be temperature-dependent with a Q₁₀ (20–30°) of 3.6 and 6.6, respectively and a Q₁₀ (30–40°) of 1.3 and 1.0, respectively. Inhibition of acetylcholinesterase by paraoxon increases to 92% the proportion of the Ch⁺ taken up which is converted to ACh. ACh did not reduce Ch⁺ transport when present at 100 μM.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

H-Pro-[3H]Leu-Gly-NH2: Uptake and Metabolism in Rat Brain

The uptake and metabolism of H-Pro-[3H]Leu-Gly-NH2 ([3H]PLG) in rat brain was investigated by reverse-phase paired-ion high pressure liquid chromatography. Following in vitro incubation of [3H]PLG with rat brain subcellular preparations, the microsomal-cytosol fraction was about twice as active in degrading PLG as the crude mitochondrial-synaptosomal fraction. For both enzyme preparations the pH optimum was found at pH 7-7.5. The major labeled metabolite was [3H]leucine, whereas 3H]labeled Leu-Gly-NH2 as the only labeled peptide intermediate was found in trace amounts. After intravenous injection of [3H]PLG the uptake of unmetabolized peptide in the brain appeared to be very low: 0.008% and 0.001% of the administered dose/g tissue at 2 and 5 min after injection respectively, while at longer survival times intact peptide was below the detection limit. Compared with the intravenous route of administration, intracerebroventricular injection of [3H]PLG yielded much higher brain concentrations of unmetabolized PLG. Following both routes of administration, the metabolite profile was in agreement with that obtained after in vitro incubation. However, the in vivo experiments also showed considerable incorporation of [3H]leucine liberated from [3H]PLG into proteins. Both the in vitro and in vivo results indicate that the initial cleavage of PLG in rat brain occurs at the NH2-terminus and that the dipeptide intermediate H-Leu-Gly-NH2 is subsequently hydrolyzed to its constituent amino acids very rapidly.