Lobeline Displaces [3H]Dihydrotetrabenazine Binding and Releases [3H]Dopamine from Rat Striatal Synaptic Vesicles: Comparison with d-Amphetamine

Abstract: Lobeline, an alkaloid from Indian tobacco (Lobelia inflata), is classified as a nicotinic agonist and is currently used as a smoking cessation agent. However, our previous in vitro studies demonstrate that lobeline does not act as a nicotinic agonist but alters presynaptic dopamine (DA) storage by potently inhibiting DA uptake into synaptic vesicles. Recently, d-amphetamine has been reported to act at the level of the synaptic vesicle to alter presynaptic function. The present in vitro studies further elucidate the mechanism of lobeline's action and compare its effects with those of d-amphetamine. [³H]Dihydrotetrabenazine ([³H]DTBZ), used routinely to probe a high-affinity binding site on the vesicular monoamine transporter (VMAT2), bound to vesicle membranes from rat striatum with a K_D of 1.67 nM and B_max of 8.68 pmol/mg of protein. Lobeline inhibited [³H]DTBZ binding with an IC₅₀ of 0.90 µM, consistent with its previously reported IC₅₀ of 0.88 µM for inhibition of [³H]DA uptake into vesicles. These results suggest that lobeline specifically interacts with DTBZ sites on VMAT2 to inhibit DA uptake into synaptic vesicles. Interestingly, d-amphetamine inhibited [³H]DTBZ binding to vesicle membranes with an IC₅₀ of 39.4 µM, a concentration 20 times greater than reported for inhibition of VMAT2 function, suggesting that d-amphetamine interacts with a different site than lobeline on VMAT2 to inhibit monoamine uptake. Kinetic analysis of [³H]DA release from [³H]DA-preloaded synaptic vesicles in the absence of drug revealed a t_1/2 of 2.12 min. Lobeline and d-amphetamine evoked [³H]DA release with EC₅₀ values of 25.3 and 2.22 µM, respectively. At a concentration 10 times the EC₅₀, lobeline and d-amphetamine significantly decreased the t_1/2 of [³H]DA release to 1.58 and 1.48 min, respectively. Thus, in contrast to d-amphetamine, which is equipotent in inhibiting DA uptake and promoting release from the synaptic vesicles, lobeline more potently (28-fold) inhibits DA uptake (via an interaction with the DTBZ site on VMAT2) than it evokes DA release to redistribute presynaptic DA storage.     

Synthesis and biological evaluation of 3-alkyl-dihydrotetrabenazine derivatives as vesicular monoamine transporter-2 (VMAT2) ligands

In the search of new probes for in vivo brain imaging of vesicular monoamine transporter type 2 (VMAT2), we have developed an efficient synthesis of a novel series of 3-alkyl-dihydrotetrabenazine (DTBZ) derivatives. The affinity of VMAT2 was evaluated by an in vitro inhibitory binding assay using [¹²⁵I]-iodovinyl-TBZ or [¹⁸F](+)-FP-DTBZ as radioligands in rat striatal tissue homogenates. New DTBZ derivatives exhibited moderate to good binding affinity to VMAT2. Among these new ligands, compound 4b showed the best affinity for VMAT2 (K_i = 5.98 nM) and may be a useful lead compound for future structure-activity studies.     

Increased MPTP Neurotoxicity in Vesicular Monoamine Transporter 2 Heterozygote Knockout Mice

Abstract: The neurotoxic action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been proposed to be attenuated by sequestration into intracellular vesicles by the vesicular monoamine transporter (VMAT2). The purpose of this study was to determine if mice with genetically reduced levels of VMAT2 (heterozygote knockout; VMAT2 +/−) were more vulnerable to MPTP. Striatal dopamine (DA) content, the levels of DA transporter (DAT) protein, and the expression of glial fibrillary acidic protein (GFAP) mRNA, a marker of gliosis, were assessed as markers of MPTP neurotoxicity. In all parameters measured VMAT2 +/− mice were more sensitive than their wild-type littermates (VMAT2 +/+). Administration of MPTP (7.5, 15, or 30 mg/kg, b.i.d.) resulted in dose-dependent reductions in striatal DA levels in both VMAT2 +/− and VMAT2 +/+ animals, but the neurotoxic potency of MPTP was approximately doubled in the VMAT2 +/− mice: 59 versus 23% DA loss 7 days after 7.5 mg/kg dose for VMAT2 +/− and VMAT2 +/+ mice, respectively. Dopaminergic nerve terminal integrity, as assessed by DAT protein expression, also revealed more drastic reductions in the VMAT2 +/− mice: 59 versus 35% loss at 7.5 mg/kg and 95 versus 58% loss at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. Expression of GFAP mRNA 2 days after MPTP was higher in the VMAT2 +/− mice than in the wild-type: 15.8- versus 7.8-fold increase at 7.5 mg/kg and 20.1- versus 9.6-fold at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. These observations clearly demonstrate that VMAT2 +/− mice are more susceptible to the neurotoxic effects of MPTP, suggesting that VMAT2-mediated sequestration of the neurotoxin into vesicles may play an important role in attenuating MPTP toxicity in vivo.     

Fluoroethoxy-1,4-diphenethylpiperidine and piperazine derivatives: Potent and selective inhibitors of [3H]dopamine uptake at the vesicular monoamine transporter-2

A small library of fluoroethoxy-1,4-diphenethyl piperidine and fluoroethoxy-1,4-diphenethyl piperazine derivatives were designed, synthesized and evaluated for their ability to inhibit [³H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and dopamine transporter (DAT), [³H]serotonin (5-HT) uptake at the serotonin transporter (SERT), and [³H]dofetilide binding at the human-ether-a-go-go-related gene (hERG) channel. The majority of the compounds exhibited potent inhibition of [³H]DA uptake at VMAT2, Ki changes in the nanomolar range (K_i?=?0.014–0.073?µM). Compound 15d exhibited the highest affinity (K_i?=?0.014?µM) at VMAT2, and had 160-, 5-, and 60-fold greater selectivity for VMAT2 vs. DAT, SERT and hERG, respectively. Compound 15b exhibited the greatest selectivity (>60-fold) for VMAT2 relative to all the other targets evaluated, and 15b had high affinity for VMAT2 (K_i?=?0.073?µM). Compound 15b was considered the lead compound from this analog series due to its high affinity and selectivity for VMAT2.     

[3H]Dihydrotetrabenazine, a New In Vitro Monoaminergic Probe for Human Brain

The monoamine transporter of dopamine (DA), noradrenaline, and 5-hydroxytryptamine synaptic vesicles was assayed in rat and human brain homogenates by in vitro binding of [3H]dihydrotetrabenazine. [3H]Reserpine, a second ligand of the vesicular monoamine transporter, could not be used. [3H]Dihydrotetrabenazine binding in rat brain was stable after 72 h at 22 degrees C postmortem. In major human brain regions, [3H]dihydrotetrabenazine binding was specific and saturable (KD, 2.7 nM). Displacement constants by substrates or inhibitors of vesicular monoamine uptake, and regional distribution in human brain were similar to those found in rodents. The highest densities of binding sites were observed in caudate nucleus, putamen, and accumbens nucleus. In caudate nucleus and in putamen from normal human subjects, [3H]dihydrotetrabenazine binding and homovanillic acid concentration were significantly or nearly significantly correlated. A weaker correlation was found between [3H]dihydrotetrabenazine binding and DA, in association with a higher variability of DA. [3H]Dihydrotetrabenazine binding in caudate nucleus and in putamen decreased significantly with age, unlike DA and homovanillic acid concentrations. The results establish [3H]dihydrotetrabenazine as a presynaptic monoaminergic ligand of interest for studies on postmortem human brain.     

Pregnenolone sulfate induces NMDA receptor dependent release of dopamine from synaptic terminals in the striatum

Neuromodulators that alter the balance between lower-frequency glutamate-mediated excitatory and higher-frequency GABA-mediated inhibitory synaptic transmission are likely to participate in core mechanisms for CNS function and may contribute to the pathophysiology of neurological disorders such as schizophrenia and Alzheimer's disease. Pregnenolone sulfate (PS) modulates both ionotropic glutamate and GABA(A) receptor mediated synaptic transmission. The enzymes necessary for PS synthesis and degradation are found in brain tissue of several species including human and rat, and up to 5 nM PS has been detected in extracts of postmortem human brain. Here, we ask whether PS could modulate transmitter release from nerve terminals located in the striatum. Superfusion of a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes with brief-duration (2 min) pulses of 25 nM PS demonstrates that PS increases the release of newly accumulated [3H]dopamine ([3H]DA), but not [14C]glutamate or [3H]GABA, whereas pregnenolone is without effect. PS does not affect dopamine transporter (DAT) mediated uptake of [3H]DA, demonstrating that it specifically affects the transmitter release mechanism. The PS-induced [3H]DA release occurs via an NMDA receptor (NMDAR) dependent mechanism as it is blocked by D-2-amino-5-phosphonovaleric acid. PS modulates DA release with very high potency, significantly increasing [3H]DA release at PS concentrations as low as 25 pM. This first report of a selective direct enhancement of synaptosomal dopamine release by PS at picomolar concentrations via an NMDAR dependent mechanism raises the possibility that dopaminergic axon terminals may be a site of action for this neurosteroid.     

Synthesis and evaluation of 2-amino-dihydrotetrabenzine derivatives as probes for imaging vesicular monoamine transporter-2

A novel series of analogs of 2-amino-dihydrotetrabenazine derivatives, 4–6, targeting the vesicular monoamine transporter have been prepared. In vitro binding was carried out in tissue homogenates prepared from rat striatal tissue homogenates with both [¹²⁵I]-iodovinyl-TBZ and [³H]DTBZ. There was a good correlation (r² = 0.925) between the affinities of the different compounds for [¹²⁵I]-iodovinyl-TBZ and [³H]-DTBZ binding. Compound 5 exhibited a better affinity for the vesicular monoamine transporter (K_i = 8.68 ± 1.26 nM and 7.01 ± 0.07 nM, respectively), which may be a good lead compound for further structural modification to develop useful probes for VMAT2.     

Putative cocaine receptor in striatum is a glycoprotein with active thiol function

Dopamine transporters of bovine and rat striata were identified by their specific [3H]cocaine binding and cocaine-sensitive [3H]dopamine [( 3H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes. Inorganic and organic mercury reagents inhibited both [3H]DA uptake and [3H]cocaine binding, though they were all more potent inhibitors of the former. n-Ethylmaleimide inhibited [3H]DA uptake totally but [3H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [3H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [3H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (less than 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (greater than 10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation.     

Characterization of high-affinity [3H]TBZOH binding to the human platelet vesicular monoamine transporter.

The present study indicates that human platelets can be used as an accessible peripheral model not only for the plasma membrane serotonin transporter, but also for the vesicular monoamine transporter. The vesicular monoamine transporter (VMAT2) is responsible for the accumulation of monoamines in the synaptic vesicles. VMAT2 differs from the plasma membrane transporters in its capability to recognize serotonin, histamine, norepinephrine and dopamine with almost the same affinity. Dihydrotetrabenazine (TBZOH) is a very potent inhibitor of VMAT2 that binds with high affinity to this transporter. [3H]TBZOH has been used as a ligand to label VMAT2 in human, bovine and rodent brain. In this study we characterized the pharmacodynamic and pharmacokinetic parameters of [3H]TBZOH binding in human platelets as compared to rat brain. The density (Bmax) and affinity (Kd) of [3H]TBZOH specific binding was assessed by Scatchard analysis. Association and dissociation rate constants (k(on), K(off)) were assessed by kinetic binding studies. In this study high-affinity and saturable binding sites for [3H]TBZOH were demonstrated in human platelets. Both the affinity of [3H]TBZOH to its binding site in platelets (Kd = 3.2+/-0.5 nM) and the kinetic rate constants (K(on) = 2.8 x 10(7) M(-1) min(-1); K(off) = 0.099 min(-1)) were similar to that in rat brain (Kd(striatum) = 1.5 nM; Kd(cerebral cortex) = 1.35 nM; K(on) = 2 x 10(7) M(-1) min(-1); K(off) = 0.069 min(-1)). Only the VMAT2 blockers tetrabenazine and reserpine inhibited [3H]TBZOH specific binding.     

Synthesis and evaluation of a series of homologues of lobelane at the vesicular monoamine transporter-2

A series of lobelane homologues has been synthesized and evaluated for their [(3)H]DTBZ binding affinity at the vesicular monoamine transporter-2 (VMAT2). The structure-activity relationships (SAR) indicate that for retention of binding affinity at VMAT2, the lengths of the methylene linkers should be no shorter than one methylene unit at C-6 of the piperidine ring, and no shorter than two methylene units at C-2 of the piperidine ring. These results indicate that the intramolecular distances between the piperidine ring and two phenyl rings in lobelane analogues are an important criterion for retention of high affinity at VMAT2.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

[3H]WIN 35,065–2: A Ligand for Cocaine Receptors in Striatum

[3H]WIN 35,065-2 binding to striatal membranes was characterized, primarily by centrifugation assay. Like [3H]cocaine, [3H]WIN 35,065-2 binds to both high- and low-affinity sites. [3H]WIN 35,065-2, however, exhibits consistently higher affinities than [3H]cocaine. Saturation experiments indicate a low-affinity binding site with an apparent KD of approximately 160 nM and a Bmax of 135 fmol/mg of tissue. A high-affinity site has also been identified with an apparent KD of 5.6 nM and a Bmax of 5.2 fmol/mg of tissue. The specific-to-nonspecific binding ratios with [3H]WIN 35,065-2 were higher than with [3H]cocaine in both centrifugation and filtration assays. Pharmacological characterization suggests that [3H]WIN 35,065-2 binds to the dopamine transporter. Mazindol, GBR 12909, nomifensine, and (-)-cocaine are potent inhibitors of [3H]WIN 35,065-2 binding. In contrast, the norepinephrine transporter ligand desipramine is a weak inhibitor, and the serotonin transporter ligand citalopram does not inhibit binding. The effect of sodium on binding was examined under conditions in which (a) the low-affinity site was primarily (87%) occupied and (b) approximately 50% of both sites were occupied. The results indicate that both sites are sodium dependent. Injection of 6-hydroxydopamine into the striatum results in a significant loss of both high- and low-affinity sites, a finding suggesting that both sites are on dopaminergic nerve terminals. Taken together, these data are consistent with the presence of multiple cocaine binding sites associated with the dopamine transporter.     

A rapid oxidation and persistent decrease in the vesicular monoamine transporter 2 after methamphetamine

Methamphetamine (METH) produces long-term decreases in markers of dopamine (DA) terminals in animals and humans. A decrease in the function of the vesicular monoamine transporter 2 (VMAT2) has been associated with damage to striatal DA terminals caused by METH; however, a possible mechanism for this decrease in VMAT2 function has not been defined. The current study showed that METH caused a rapid decrease to 68% of controls in VMAT2 protein immunoreactivity of the vesicular fraction from striatal synaptosomes within 1 h after a repeated high-dose administration regimen of METH. This decrease was associated with a 75% increase in nitrosylation of VMAT2 protein in the synaptosomal fraction as measured by nitrosocysteine immunoreactivity of VMAT2 protein. The rapid decreases in VMAT2 persisted when evaluated 7 days later and were illustrated by decreases in VMAT2 immunoreactivity and DA content of the vesicular fraction to 34% and 51% of control values, respectively. The decreases were blocked or attenuated by prior injections of the neuronal nitric oxide synthase inhibitor, S-methyl-l-thiocitrulline. These studies demonstrate that METH causes a rapid neuronal nitric oxide synthase-dependent oxidation of VMAT2 and long-term decreases in VMAT2 protein and function. The results also suggest that surviving DA terminals after METH exposure may have a compromised capacity to buffer cytosolic DA concentrations and DA-derived oxidative stress.     

The Transport Systems for Selenomethionine/Methionine and Selenocystine/Cystine in Escherichia Coli K-12 Appear to be Cooperative

Dopamine transporters of bovine and rat striata were identified by their specific [³H]cocaine binding and cocaine-sensitive [³H]dopamine ([³H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes

Inorganic and organic mercury reagents inhibited both [³H]DA uptake and [³H]cocaine binding, though they were all more potent inhibitors of the former, n- Ethylmaleimide inhibited [³H]DA uptake totally but [³H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [³H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [³H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (< 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (>10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation     

Reduced vesicular storage of dopamine exacerbates methamphetamine-induced neurodegeneration and astrogliosis

The vesicular monoamine transporter 2 (VMAT2) controls the loading of dopamine (DA) into vesicles and therefore determines synaptic properties such as quantal size, receptor sensitivity, and vesicular and cytosolic DA concentration. Impairment of proper DA compartmentalization is postulated to underlie the sensitivity of DA neurons to oxidative damage and degeneration. It is known that DA can auto-oxidize in the cytosol to form quinones and other oxidative species and that this production of oxidative stress is thought to be a critical factor in DA terminal loss after methamphetamine (METH) exposure. Using a mutant strain of mice (VMAT2 LO), which have only 5–10% of the VMAT2 expressed by wild-type animals, we show that VMAT2 is a major determinant of METH toxicity in the striatum. Subsequent to METH exposure, the VMAT2 LO mice show an exacerbated loss of dopamine transporter and tyrosine hydroxylase (TH), as well as enhanced astrogliosis and protein carbonyl formation. More importantly, VMAT2 LO mice show massive argyrophilic deposits in the striatum after METH, indicating that VMAT2 is a regulator of METH-induced neurodegeneration. The increased METH neurotoxicity in VMAT2 LO occurs in the absence of any significant difference in basal temperature or METH-induced hyperthermia. Furthermore, primary midbrain cultures from VMAT2 LO mice show more oxidative stress generation and a greater loss of TH positive processes than wild-type cultures after METH exposure. Elevated markers of neurotoxicity in VMAT2 LO mice and cultures suggest that the capacity to store DA determines the amount of oxidative stress and neurodegeneration after METH administration.     

Physiological presynaptic facilitation of dopamine release in the cat caudate nucleus

Using halothane-anaesthetized cats implanted with push-pull cannulae, we investigated the effects of GABA application into the VM/VL on the release of [3H] DA continuously synthetized from [3H] tyrosine in the ipsilateral CN and SN and on single unit activity of nigral DA cells. GABA was applied (30 min) at a concentration of 10(-3) or 10(-5) M since the higher concentration reduces the local multi-unit activity in the VM/VL while the opposite response is observed with the lower one. The application of GABA into the VM/VL at a concentration of 10(-3) M resulted in an increase in nigral [3H] DA release, an inhibition of DA cell firing and a decrease in [3H] DA release in the CN. The latter effect is likely due to the inhibition of DA neuron activity which is triggered through DA autoreceptors by DA released from dendrites. In contrast, when applied at a concentration of 10(-5) M into the VM/VL, GABA stimulated [3H] DA release in the CN despite its inhibitory effect on single unit activity of DA cells. Furthermore, the nigral release of [3H] DA was no longer affected. These results indicated that DA release from nerve terminals was not dependent on nerve activity and they favour the existence of a potent facilitatory presynaptic regulation of DA release. The intervention of a presynaptic mechanism was further established by examining the effect of GABA (10(-5) M) application into the VM/VL on [3H] DA release in the CN shortly after a complete ipsilateral hemisection of the brain made at the meso-diencephalic level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of vesicular uptake of monoamines by hyperforin

Hyperforin is the major active ingredient of Hypericum perforatum (St John's Wort), a traditional antidepressant medication. This study evaluated its inhibitory effects on the synaptic uptake of monoamines in rat forebrain homogenates, comparing the nature of the inhibition at synaptic and vesicular monoamine transporters. A hyperforin-rich extract inhibited with equal potencies the sodium-dependent uptake of the monoamine neurotransmitters serotonin [5-HT], dopamine [DA] and norepinephrine [NE] into rat brain synaptosomes. Hyperforin inhibited the uptake of all three monoamines noncompetitively, in marked contrast with the competitive inhibition exerted by fluoxetine, GBR12909 or desipramine on the uptake of these monoamines. Hyperforin had no inhibitory effect on the binding of [3H]paroxetine, [3H]GBR12935 and [3H]nisoxetine to membrane presynaptic transporters for 5-HT, DA and NE, respectively. The apparent presynaptic inhibition of monoamine uptake could reflect a "reserpine-like mechanism" by which hyperforin induced release of neurotransmitters from synaptic vesicles into the cytoplasm. Thus, we assessed the effects of hyperforin on the vesicular monoamine transporter. Hyperforin inhibited with equal potencies the uptake of the three tritiated monoamines to rat brain synaptic vesicles. Similarly to the synaptosomal uptake, the vesicular uptake was also noncompetitively inhibited by hyperforin. Notably, hyperforin did not affect the direct binding on [3H]dihydrotetrabenazine, a selective vesicular monoamine transporter ligand, to rat forebrain membranes. Our results support the notion that hyperforin interferes with the storage of monoamines in synaptic vesicles, rather than being a selective inhibitor of either synaptic membrane or vesicular monoamine transporters.     

Nicotinic Modulation of [3H]Dopamine Release from Striatal Synaptosomes: Pharmacological Characterisation

Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. We have compared the effects of a number of nicotinic agonists and antagonists on a perfused synaptosome preparation preloaded with [3H]dopamine. (-)-Nicotine, acetylcholine, and the nicotinic agonists cytisine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), at micromolar concentrations, stimulated the release of [3H]dopamine from striatal nerve terminals. Carbamylcholine was a much weaker agonist. The actions of (-)-nicotine, cytisine, and DMPP were inhibited by low concentrations of the nicotinic antagonists dihydro-beta-erythroidine, mecamylamine, pempidine, and neosurugatoxin; alpha-bungarotoxin was without effect, and extending the time of exposure to this toxin resulted in only very modest inhibition. This pharmacology points to a specific nicotinic receptor mechanism that is clearly distinct from that at the neuromuscular junction. Atropine failed to antagonise the effects of acetylcholine and carbamylcholine, suggesting that no muscarinic component is involved. The nicotinic receptor ligands (-)-[3H]nicotine and 125I-alpha-bungarotoxin bound to specific sites enriched in the synaptosome preparation. Drugs tested on the perfused synaptosomes were examined for their ability to interact with these two ligand binding sites in brain membranes. The differential sensitivity to the neurotoxins alpha-bungarotoxin and neosurugatoxin of the 125I-alpha-bungarotoxin and (-)-[3H]nicotine binding sites, respectively, leads to a tentative correlation of the (-)-[3H]nicotine site with the presynaptic nicotinic receptor on striatal nerve terminals.     

21-Aminosteroids Interact with the Dopamine Transporter to Protect Against 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

U-78518F, a 21-aminosteroid from the novel family of lipid peroxidation inhibitors (lazaroids), increased survival of dopamine (DA) neurons in mesencephalic cell cultures incubated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Protection against DA neuron death occurred with increasing concentrations of U-78518F up to 30 microM. Non-specific toxicity produced with higher concentrations of MPP+ was not affected by the lazaroid. U-78518F inhibited cellular uptake of [3H]MPP+ and [3H]DA, but not that of gamma-[3H]aminobutyric acid. In human striatal membrane preparations, U-78518F competed with [3H]mazindol for binding to the DA transporter, with a calculated Ki value of 10 microM. Two of four lazaroids tested inhibited [3H]DA uptake in the cell culture system. The protective effects of 21-aminosteroids in MPP(+)-induced neurotoxicity are, in part, a function of the interaction of these agents with the DA transporter.     

Subcellular Site of Action of Imipramine in Rodent Brain

To determine the site of action of imipramine, the subcellular distribution of [3H]imipramine in rodents was followed after both in vivo administration and in vitro incubation with tissue slices under "physiological" conditions. Total [3H]imipramine (10-1,000 nM) binding was associated with all primary fractions, but in particular with the nuclear (P1) and mitochondrial (P2) pellets and the synaptosomal (P2B) and myelin (P2A) fractions. Using an excess of imipramine to define any nonspecific interactions, a specific association was observed mainly in those fractions containing isolated nerve terminals and to a lesser extent with the purified myelin fraction. Preparation of subsynaptosomal fractions by osmotic lysis indicated that [3H]imipramine was associated with the synaptic vesicle and microsomal fractions and also with synaptosomal membranes. The degree of binding to the vesicular and microsomal fractions was increased with the length of preparation time, whereas there was an inverse relationship between the length of preparation and the amount bound to the synaptosomal membrane fraction. There was no evidence of an intrasynaptosomal accumulation of [3H]imipramine at concentrations up to 1,000 nM. [3H]2-Nitroimipramine, a slowly dissociating imipramine derivative, was exclusively located in synaptic membrane fractions. Prior treatment of rats with a combination of 5,7-dihydroxytryptamine and desipramine reduced 5-hydroxytryptamine levels and the levels of [3H]imipramine associated with the synaptosomal fractions to the same extent. It is concluded that imipramine is associated with a binding site localised on 5-hydroxytryptaminergic nerve terminals and that there is a redistribution to other sites (vesicular and microsomal) during the isolation procedure.