Striatal Synaptosomal Preparations Research Articles

Identification of a Novel Allosteric Modulator of the Human Dopamine Transporter.

The dopamine transporter (DAT) serves a pivotal role in controlling dopamine (DA)-mediated neurotransmission by clearing DA from synaptic and perisynaptic spaces and controlling its action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DAT to mediate their effects by enhancing extracellular DA concentrations. We previously identified a novel allosteric site in the related human serotonin transporter that lies outside the central substrate and inhibitor binding pocket. We used the hybrid structure based (HSB) method to screen for allosteric modulator molecules that target a similar site in DAT. We identified a compound, KM822, that was found to be a selective, noncompetitive inhibitor of DAT. We confirmed the structural determinants of KM822 allosteric binding within the allosteric site by structure/function and substituted cysteine scanning accessibility biotinylation experiments. In the in vitro cell-based assay and ex vivo in both rat striatal synaptosomal and slice preparations, KM822 was found to decrease the affinity of cocaine for DAT. The in vivo effects of KM822 on cocaine were tested on psychostimulant-associated behaviors in a planarian model where KM822 specifically inhibited the locomotion elicited by DAT-interacting stimulants amphetamine and cocaine. Overall, KM822 provides a unique opportunity as a molecular probe to examine allosteric modulation of DAT function.

Effectiveness of Nicotinic Agonists as Desensitizers at Presynaptic 4 2- and 4 5 2-Nicotinic Acetylcholine Receptors

Nicotine interacts with nicotinic acetylcholine receptors (nAChRs) and modifies neuronal functions. The net result of nicotine exposure is difficult to assess because multiple nAChR subtypes exist and are expressed on multiple classes of neurons. Nicotine, unlike the natural agonist acetylcholine, remains in tissues for hours, and during this extended exposure nAChRs desensitize. Therefore, agonists can block the natural functions of nAChRs. Higher nicotine concentrations are required to desensitize α4β2-nAChRs containing the α5 subunit. The aim of these experiments was to determine if this property holds true for compounds other than nicotine. [(3)H]-dopamine release from crude mouse striatal synaptosomal preparations was used to measure activation and desensitization of the [(α4β2)2β2] and [(α4β2)2α5] nAChR subtypes. Affinity was measured by competition with [(125)I]-epibatidine. Nine compounds of varying affinity and efficacy were tested. All compounds partially desensitized both subtypes; concentration necessary for desensitization correlated with binding site affinity but not efficacy. All compounds showed a similar, significant shift in concentration necessary for a 50% effect when the α5 subunit was included (averaging 8-fold higher). The extent of desensitization produced by a 10-min exposure did not correlate with affinity or efficacy of compound. Full or partial nicotinic agonists used as medications may effectively desensitize α4β2-nAChRs. However, significantly higher concentrations of all compounds tested were required to elicit desensitization of α4α5β2-nAChRs than α4β2-nAChRs. If desensitization is the important property for a smoking cessation drug, basic screening at both subtypes may provide a mechanistic foundation for effectiveness.

Ex vivo identification of protein–protein interactions involving the dopamine transporter

The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT–protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca 2+/calmodulin-dependent protein kinase II (CaMKII) β, CaMKII δ, protein kinase C (PKC) β, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein–protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein–protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT.

The PKC inhibitor Ro31-8220 blocks acute amphetamine-induced dopamine overflow in the nucleus accumbens

Acute administration of the psychostimulant amphetamine increases extracellular levels of dopamine (DA) by reversing the DA transporter on ascending midbrain DA neurons. In vitro studies using striatal synaptosomal, slice and nucleus accumbens (NAcc) tissue preparations have implicated protein kinase C (PKC) in this effect. The present study further examined this effect in vivo by assessing the ability of the PKC inhibitor, Ro31-8220 (10 μM), to inhibit acute amphetamine-induced DA overflow when applied with this drug to the NAcc via reverse dialysis. Amphetamine was applied at a concentration of 30 μM, and the core and shell subregions of the NAcc were assayed separately in freely moving rats. These brain regions play a role in the acute locomotor-activating and motivational effects of amphetamine. Consistent with the findings of previous in vitro experiments, reverse dialysis of Ro31-8220 with amphetamine robustly attenuated the ability of this drug to increase extracellular levels of dopamine in both the core and shell subregions of the NAcc. These results confirm that amphetamine stimulates dopamine overflow via a PKC-dependent mechanism.

Effect of lead exposure on dopaminergic transmission in the rat brain

Lead is a neurotoxicant with known behavioral and neurochemical effects. In this study we attempted to relate the behavioral effects of lead to neurotransmission. Oral administration of 1000 ppm of lead acetate to young rats for 30 days caused a reduction in locomotor activity and stereotypic exploratory behavior during a 20 min testing period. This locomotor hypoactivity induced by lead was accompanied by a reduction in stereotypic behavior (sniffing, lickings, biting and grooming). These outcomes suggested that lead might interfere with catecholaminergic and particularly dopaminergic neurotransmission. Therefore, we examined the effect of the lead acetate on the uptake of dopamine in striatal synaptosomal preparations. The collected data showed a clear inhibition of the uptake of 3H-DA with an IC50 of 3.5 x 10(-5)M. This inhibition of the uptake of dopamine suggests that the behavioral effects of lead may be involved in dopaminergic neurotransmission.

Substrate-induced trafficking of the dopamine transporter in heterologously expressing cells and in rat striatal synaptosomal preparations.

Dopamine transporter (DAT) trafficking was assessed by functional measurements of dopamine uptake and by biotinylation of surface proteins followed by gel electrophoresis and Western blotting. In human embryonic kidney (HEK)-293 cells expressing human DAT (HEK-hDAT), pretreatment with dopamine (0.1-100 microM) followed by washout caused reductions in subsequent dopamine uptake (reflected in Vmax) with effective dopamine concentrations in the 10 to 100 microM range and pretreatment times of 10 to 60 min. Reductions assessed after 60-min pretreatment with 100 microM dopamine corresponded with decreases measured in surface DAT by the noncleavable biotin method, which were caused, at least in part, by enhanced endocytosis as monitored with cleavable biotin. Pretreatment of rat striatal synaptosomes with dopamine (10 and 100 microM) also caused reductions in DAT uptake activity (Vmax), and again the underlying mechanism seemed to be a diminished presence of DAT at the surface of synaptosomes as measured by the noncleavable biotin method. The copresence of cocaine during pretreatment with dopamine prevented the down-regulation of surface DAT. The present results show that DAT surface residency can be regulated by substrate acting on it, not only in cells heterologously expressing DAT but also in situ in rat brain tissue.

395 Embryotoxicitic effects of ranitidine

Acute administration of the agricultural fungicide triadimefon produced a neurotoxic syndrome in rats characterized by increased motor activity, stereotyped behaviors, and altered monoamine metabolism. Triadimenol, a metabolite of triadimefon in mammals, plants, and soil, also increased motor activity in rodents. To test the hypothesis that triadimefon and triadimenol are indirect-acting dopamine agonists, the present studies examined their abilities to inhibit monoamine uptake, bind to the dopamine transporter, and stimulate dopamine efflux in rat brain tissue,in vitro.Both triazoles inhibited the uptake of dopamine in striatal synaptosomal preparations. Triadimefon was 100-fold less potent than GBR12909, a prototypical inhibitor of dopamine uptake (IC50 = 4.7 μmvs. 37.2 nm, respectively), and triadimenol was about threefold less potent than triadimefon. Triadimefon also weakly inhibited the uptake of norepinephrine in cortical synaptosomes (IC50 = 22.4 μm), but neither compound blocked the uptake of serotonin in cortical synaptosomes (IC50s > 100 μm). Triadimefon and triadimenol had similar affinity for [3H]mazindol binding sites on the dopamine transporter (IC50s ≈ 1–1.5 μm, only two- to threefold greater than GBR12909). Neither triadimefon nor triadimenol (0.01–100 μm) increased basal efflux of [3H]DA that had been preloaded into striatal mincesin vitro.An unexpected result was that GBR12909 (10 μm) increased basal efflux of [3H]DA by 71%, suggesting that this compound has DA releasing properties. These data suggest that increased synaptic concentrations of dopamine due to inhibition of dopamine uptake may play an important role in the neurobehavioral effects of triadimefon and triadimenol.

Serotonin depletion produces long lasting increase in striatal glutamatergic transmission

The ability of serotonin (5-HT) to influence striatal glutamatergic transmission was examined by determining changes over time in glutamate extracellular levels, transporter expression and synaptosomal uptake in rats with lesion of serotonergic neurones. By 8 days after intraraphe injections of 5,7-dihydroxytryptamine, producing 80% decreases in striatal tissue 5-HT levels, no changes were observed in the glutamatergic transmission. When 5-HT depletion was almost complete (21 days post-lesion), high affinity glutamate uptake in striatal synaptosomal preparations was significantly increased (156% of control), although no changes in striatal GLT1, GLAST and EAAC1 mRNAs, and GLT1 protein were detected by in situ hybridization and immunohistochemistry. Meanwhile, the serotonin lesion produced large increases in basal extracellular levels of glutamate and glutamine (364% and 259%, respectively) determined in awake rats by in vivo microdialysis, whereas no change was observed in dopamine levels as compared with control rats. High potassium depolarization as well as L-trans-pyrrolidine-2,4-dicarboxylate, also induced larger increases in extracellular levels of glutamate in lesioned rats than in controls. Finally, similar changes in glutamate transmission were observed by 3 months post-lesion. These results suggest that 5-HT has a long lasting and tonic inhibitory influence on the striatal glutamatergic input, without affecting the basal dopaminergic transmission.

P-Fluorophenylglycine in the urine of baboons treated with HPTP, the tetrahydropyridine analog of haloperidol

We report the presence of p-fluorophenylglycine ( p-FPG) in the urine of six baboons treated with HPTP, the tetrahydropyridine dehydration product of haloperidol (HP). Oxidative N-dealkylation, the major metabolic pathway of HP, gives rise to 3-(4-fluorobenzoyl)propionic acid ( p-FBPA). Subsequent β-oxidation of p-FBPA produces p-fluorophenylacetic acid ( p-FPA). The presence of p-FPA argues for the formation also of p-fluorophenylglyoxylic acid ( p-FPGA) derived from β-oxidation of p-FBPA. Plasma aminotransferases should convert p-FPGA to p-FPG. The presence of p-FPG in these animals suggest the presence of phenylglycine aminotransferases in the baboon and possibly also in other primates, including the human. Reports by other authors found that treatment with α-phenylglycine (α-PG), an “unnatural” amino acid, leads to striatal dopamine (DA) depletion in rabbits — an effect explained on the basis of α-PG competing with DA for the neuronal vesicular storage sites. We performed in vitro DA release assays in mouse striatal synaptosomal preparations but found that neither α-PG nor p-FPG released any DA. It therefore remains unclear whether p-FPG may be a contributing factor to neurologic side-effects such as tardive dyskinesia (TD) found in patients after long-term HP treatment.

17] Inhibition of [3H]dopamine translocation and [3H]cocaine analog binding: A potential screening device for cocaine antagonists

Publisher Summary This chapter presents an overview of a potential screening device for cocaine antagonists. There is considerable interest in developing a cocaine antagonist for use as an adjunct in the treatment of cocaine addiction or as a counteractive medication to combat acute central overstimulation. Because the dopamine transporter in the plasma membrane is one of the targets considered crucial for its centrally stimulatory activity, it is logical to conceive of a compound that interferes with the action of cocaine at the transporter level. Antagonism of cocaine action can be accommodated in a model depicting separate sites of action for uptake blockers and substrate recognition. The general approach in screening for a potential cocaine antagonist consists of assessing the inhibitory effect of a test compound on the translocation of dopamine by the dopamine transporter, usually in rat striatal synaptosomal preparations, as well as its inhibitory effect on the binding of a radiolabeled cocaine analog. The aim is to find a compound that potently inhibits binding without affecting dopamine uptake.

Triadimefon and Triadimenol: Effects on Monoamine Uptake and Release

Acute administration of the agricultural fungicide triadimefon produced a neurotoxic syndrome in rats characterized by increased motor activity, stereotyped behaviors, and altered monoamine metabolism. Triadimenol, a metabolite of triadimefon in mammals, plants, and soil, also increased motor activity in rodents. To test the hypothesis that triadimefon and triadimenol are indirect-acting dopamine agonists, the present studies examined their abilities to inhibit monoamine uptake, bind to the dopamine transporter, and stimulate dopamine efflux in rat brain tissue,in vitro.Both triazoles inhibited the uptake of dopamine in striatal synaptosomal preparations. Triadimefon was 100-fold less potent than GBR12909, a prototypical inhibitor of dopamine uptake (IC50 = 4.7 μmvs. 37.2 nm, respectively), and triadimenol was about threefold less potent than triadimefon. Triadimefon also weakly inhibited the uptake of norepinephrine in cortical synaptosomes (IC50 = 22.4 μm), but neither compound blocked the uptake of serotonin in cortical synaptosomes (IC50s > 100 μm). Triadimefon and triadimenol had similar affinity for [3H]mazindol binding sites on the dopamine transporter (IC50s ≈ 1–1.5 μm, only two- to threefold greater than GBR12909). Neither triadimefon nor triadimenol (0.01–100 μm) increased basal efflux of [3H]DA that had been preloaded into striatal mincesin vitro.An unexpected result was that GBR12909 (10 μm) increased basal efflux of [3H]DA by 71%, suggesting that this compound has DA releasing properties. These data suggest that increased synaptic concentrations of dopamine due to inhibition of dopamine uptake may play an important role in the neurobehavioral effects of triadimefon and triadimenol.

Metabotropic glutamate receptor agonists inhibit endogenous glutamate release from rat striatal synaptosomes

A striatal synaptosomal preparation was used to assess the action of metabotropic glutamate receptor (mGlu receptor) agonists on 4-aminopyridine (2 mM)-stimulated endogenous glutamate release. 4-Aminopyridine alone increased basal glutamate release by 6.89 ± 0.74 nmol/mg. The mGlu receptor agonists l-2-amino-4-phosphonobutyric acid ( l-AP4) (IC 50 ≈ 0.2 μM) and (1 S,3 S)-1-amino-cyclopentane-3,3-dicarboxylic acid (IC 50 ≈ 0.2 μM) inhibited 4-aminopyridine-stimulated release. The putative mGlu receptor antagonist ( S)-α-methyl- l-AP4, which itself inhibited 4-aminopyridine-stimulated release (IC 50 ≈ 10 μM), did not inhibit the effects of the two agonists.

Translocation of dopamine and binding of 2β-carbomethoxy-3 β-(4-fluorophenyl) tropane (WIN 35,428) measured under identical conditions in rat striatal synaptosomal preparations: Inhibition by various blockers

Translocation of [ 3H]dopamine and binding of 2 β-carbomethoxy-3 β-(4-fluorophenyl)[ 3H]-tropane ([ 3H]WIN 35,428) were measured in crude synaptosomal preparations from rat striatum under identical conditions of assay buffer (phosphate-Krebs) and temperature (25 °). [ 3H]Dopamine uptake as a function of time was close to linear for at least 8 min, whereas [ 3H]WIN 35,428 binding had reached equilibrium within 1 min and remained at its plateau value for at least 20 min. The following inhibitors were tested in uptake and binding assays run in parallel with the same synaptosomal preparation: cocaine, WIN 35,428, benztropine, nomifensine, mazindol, methylphenidate, ndash[1-(2-benzo[b]-thiophenyl)cyclohexyl]piperidine (BTCP), Lu 19-005 (Indatraline), 1 — (2 — (di(4 — fluorophenyl)-methoxy)-ethyl) -4-(3-phenyl-2-propyl)piperazine (GBR 12909), 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenyl-2-propyl) piperazine (GBR 12935) and 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo[1,2-a] quinoxaline (CGS 12066B). When present together with [ 3H]dopamine or [ 3H]WIN 35,428 for 8 min, the observed binding ic 50 values were generally higher (average 1.4-fold) than the uptake ic 50 values, with a significant y-axis intercept in linear regression analysis of binding on uptake ic 50. For slowly equilibrating inhibitors, estimates of uptake ic 50 values were overestimates, and relatively lower values were obtained by monitoring [ 3H]dopamine uptake for 1 min only during the last minute of the 8-min presence of inhibitor; under these conditions, binding over uptake ic 50 ratios were on the average 2.3. Kinetic calculations, taking into account both radioligand and inhibitor equilibration kinetics, indicated that the latter comparison between binding and uptake measurements was most relevant, and suggested the involvement of complexities beyond simple competitive inhibition of dopamine transport, such as different binding domains for substrate and blocker recognition, or spare receptors for blockers. The present data indicate that binding over uptake ic 50 ratios should be interpreted with caution, depending on the experimental conditions used to measure these ratios.

Dopamine and γ-aminobutyric acid transporters: Differential regulation by agents that promote phosphorylation

Treatment of striatal synaptosomes with the protein phosphatase inhibitor okadaic acid significantly decreased γ-aminobutyric acid (GABA) uptake, indicating that the GABA transporter may be regulated by phosphorylation. Forskolin and 8-bromoadenosine-3,5-cyclic monophosphate (8-br-cAMP) inhibited GABA uptake to the same extent as okadaic acid, suggesting the involvement of protein kinase A in GABA transporter regulation. In contrast, the same treatments did not alter dopamine (DA) uptake into striatal synaptosomal preparations. The results suggest that the structurally related GABA and DA transporters may be subject to different post-translational regulation.

Nitric oxide inhibits [ 3H]dopamine uptake

Sodium nitroprusside (SNP), a generator of nitric oxide (NO), decreased [ 3H]dopamine uptake into rat striatal synaptosomal preparations in a dose-, time- and temperature-dependent fashion. Reduced hemoglobin, a substance that binds NO, prevented the SNP-induced decrease in uptake. Potassium ferri- and ferrocyanide, compounds similar to SNP that do not produce NO, were without effect on uptake. SNP inhibited [ 3H]dopamine uptake in synaptosomes from nucleus accumbens and olfactory tubercle as well but with a lower potency. SNP inhibited [ 3H]serotonin and [ 3H]glutamate uptake but had no effect on [ 3H]norepinephrine uptake. S-Nitroso- N-acetylpenicillamine (SNAP), another generator of NO, had effects similar to those of SNP. The SNP-induced decrease in [ 3H]dopamine uptake was due to a V max decrease at 100 μM SNP and to both a V max and K m change at 300 μM SNP. Depletion of calcium by omission of calcium from buffers and addition of EGTA increased the potency of SNP in inhibiting uptake. There was no change in [ 3H]WIN 35428 binding to the dopamine transporter with doses of SNP that inhibited uptake. These data indicate that NO can decrease [ 3H]dopamine transporter function.

Effects of pertussis toxin on inhibition of synaptosomal tyrosine hydroxylase activity by apomorphine

The effects of pertussis toxin (PTX) on synaptosomal tyrosine hydroxylase (TH) activity and on the inhibition of synaptosomal TH activity by apomorphine were investigated. Exposure of striatal synaptosomes to PTX does not affect basal- or forskolin-stimulated TH activity, but attenuates apomorphine-elicited inhibition of forskolin-stimulated synaptosomal TH activity. There is a good correlation between the attenuation of apomorphine-elicited inhibition of synaptosomal TH activity by PTX and (−)-sulpiride, suggesting that G proteins are involved in the dopamine (DA) autoreceptor-mediated regulation of the enzyme activity. The exposure of synaptosome to PTX results in a 40–50% inactivation of G i and G o proteins, which is evident from the reduction of ADP ribosylation with [ 32P]NAD of the remaining G proteins following preincubation with the toxin. The present study also demonstrates that striatal synaptosomal preparations can be used for investigations of the molecular properties of nerve terminal DA autoreceptors.

Dopamine uptake inhibitory capacities of β-carboline and 3,4-dihydro-β-carboline analogs of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) oxidation products

Potentially endogenous β-carboline and 3,4-dihydro-β-carboline alkaloidal compounds were compared, generally as 2-methylated (quaternary) and normethylated pairs, to the neurotoxin, 1-methyl-4-phenyl-dihydropyridinium ion (MPP +), with respect to inhibition of [ 3H]dopamine uptake into rat striatal synaptosomal preparations. Although less potent than MPP +, several compounds displayed IC 50 values for inhibition in the moderate range (12–24 μM). Notably, quaternization generally did not improve inhibitory potency, and the 3,4-dihydro-compounds often were more effective inhibitors than their heteroaromatic analogs. The partially competetive nature of inhibition by one of the more effective pairs, 2-methyl-harmine and harmine, was consistent with uptake of the beta-carbolines by the synaptosomal dopamine uptake system, as was the fact that the accumulation of 2-[ 14C]methyl-harmine was significantly reduced by low Na + media and by nomifensine, a potent inhibitor of the dopamine transporter. When viewed with reports that certain 2-methyl-β-carbolines show MPP +-like toxicity in vitro and in vivo, these studies support the proposal that a mammalian β-carbolinium compound may be taken up by nigrostriatal neurons and provoke the neuronal degeneration underlying Parkinson's disease.

Mode of action of tiaspirone on the central cholinergic system

Tiaspirone, a potential antipsychotic drug, reduced the acetylcholine content of rat hemispheric brain regions (striatum 35%, hippocampus 20%, cortex 32% with no effect on N. accumbens) at an oral dose of 40 mg/kg. Choline content was uniformly raised in the same brain regions. A kinetic study showed that the drug is evenly distributed in the brain. Tiaspirone's effects on acetylcholine and choline in the striatum were not related in time. The fall off (30-240 min) of tiaspirone's effect on choline content paralleled the decline in striatal drug concentration (t1/2 = 240 min) whereas that on acetylcholine did not. No tolerance was observed to an acute challenge with tiaspirone on acetylcholine and choline in the striatum after 11 days' subchronic treatment. In vitro the drug had no effect on striatal choline acetyltransferase and acetylcholinesterase activities up to a concentration of 300 microM. The muscarinic agonist oxotremorine did not interfere with the acetylcholine decrease produced by the drug suggesting that muscarinic receptors are not essential for this effect. Tiaspirone, however, was found to be a competitive, reversible inhibitor of the sodium-dependent high-affinity choline uptake (SDHACU) by crude hippocampal and striatal synaptosomal preparations, giving IC50 values of respectively 3.69 microM and 1.14 microM. The compound did not alter SDHACU ex vivo despite the fact that it readily crosses the blood-brain barrier and achieves brain concentrations equivalent to its in vitro IC50 concentration. Tiaspirone antagonized the striatal acetylcholine increasing effect of apomorphine, a selective dopaminergic receptor agonist, supporting the idea that the drug affects the striatal cholinergic system by a primary action on dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions Between α‐Latrotoxin and Trivalent Cations in Rat Striatal Synaptosomal Preparations

The interactions between alpha-latrotoxin (alpha-LTx), a neurosecretagogue purified from the venom of the black widow spider, and the trivalent cations Al3+, Y3+, La3+, Gd3+, and Yb3+ were investigated in rat striatal synaptosomal preparations. All trivalent cations tested were inhibitors of alpha-LTx-induced [3H]dopamine [( 3H]DA) release (order of potency: Yb3+ greater than Gd3+ approximately Y3+ greater than La3+ greater than Al3+). Only with Al3+ could inhibition of [3H]DA release be attributed to a block of 125I-alpha-LTx specific binding to synaptosomal preparations. The inhibitory effect of trivalent ions was reversible provided synaptosomes were washed with buffer containing EDTA. Trivalent ions also inhibited alpha-LTx-induced [3H]DA release at times when alpha-LTx-stimulated release was already evident. alpha-LTx-induced synaptosomal membrane depolarization was blocked by La3+, but not affected by Gd3+, Y3+, and Yb3+. alpha-LTx-stimulated uptake of 45Ca2+ was inhibited by all trivalent cations tested. These results demonstrate that there exist at least three means by which trivalent cations can inhibit alpha-LTx action in rat striatal synaptosomal preparations: (1) inhibition of alpha-LTx binding (Al3+); (2) inhibition of alpha-LTx-induced depolarization (La3+); and (3) inhibition of alpha-LTx-induced 45Ca2+ uptake (Gd3+, Y3+, Yb3+, La3+).

Methylpiperidine analog of hemicholinium-3: A selective, high affinity non-competitive inhibitor of sodium dependent choline uptake system

The potency of hemicholinium-3 (HC-3) and its analogs to inhibit sodium dependent high affinity choline uptake were evaluated in rat striatal synaptosomal preparation. Hemicholinium-3 inhibited sodium dependent high affinity choline uptake (IC 50 = 18 nM) while the half molecule of HC-3, HC-15, was inactive. The order of potency for choline uptake inhibition of piperidine substituted HC-3 molecule was as follows: 4-methylpiperidine ( A-5 and CA-5) ⋙ HC-3 ⪢ unsubstituted piperidines (CA-1 and A-1) ⪢ 2- or 3- methylpiperidine (A-2 and A-3) and 4-hydroxypiperidine (A-7). The tertiary amine derivative of 4-methylpiperidine substituted HC-3 (A-4) was nearly 10-fold less potent than its corresponding quaternary derivative (A-5). Choline uptake was inhibited competitively by HC-3 and non-competitively by A-5. The inhibition of choline uptake by A-5 was readily reversible by washing. A-5 did not inhibit the uptake of dopamine and γ-aminobutyric acid. These findings suggest that the N-methyl,4-methylpiperidine analog of HC-3 (A-5) is the most potent of all known inhibitors of sodium dependent high affinity choline uptake and that the inhibition of choline uptake by this compound is mediated through a mechanism distinct from a simple competitive one.