The role of conserved tryptophan and acidic residues in the human dopamine transporter as characterized by site-directed mutagenesis

The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and acidic residues that are completely or highly conserved among Na(+)/Cl(-)-dependent transporters. We have explored the roles of these residues using non-conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184L) lacked plasma membrane immunostaining and were not functional. Both DA uptake and cocaine analog (i.e. 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative selectivity of the hDAT for cocaine analogs and DA by 10-24-fold. In particular, mutation of D345 in the third intracellular loop still allowed considerable [(3)H]DA uptake, but caused undetectable [(3)H]CFT binding. Upon anti-C-terminal-hDAT immunoblotting, D345N appeared as broad bands of 66-97 kDa, but this band could not be photoaffinity labeled with cocaine analog [(125)I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid ([(125)I]RTI-82). Unexpectedly, in this mutant, cocaine-like drugs remained potent inhibitors of [(3)H]DA uptake. CFT solely raised the K(m) of [(3)H]DA uptake in wild-type hDAT, but increased K(m) and decreased V(max) in D345N, suggesting different mechanisms of inhibition. The data taken together indicate that mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding of cocaine to the transporter may not be the only way by which cocaine analogs inhibit DA uptake.     

PI 3-kinase regulation of dopamine uptake

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3-kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [(3) H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3-kinase, increased [(3)H]DA uptake and blocked the amphetamine-induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3-kinase. The LY294002-induced reduction in [(3)H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin-dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.     

N-methyl-D-aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release display differential sensitivity to quinolinic acid, the HIV-1 envelope protein gp120, external pH and protein kinase C inhibition

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [(3)H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 microM or 1 mM) plus glycine (1 microM)-elicited [(3)H]NA, but not [(3)H]DA release. The NMDA (100 microM)-evoked release of [(3)H]NA and [(3)H]DA was similar and concentration-dependently enhanced by glycine or D-serine (0.1-1 microM); in contrast, the HIV-1 envelope protein gp120 potently (30-100 pM) enhanced the NMDA-evoked release of [(3)H]NA, but not that of [(3)H]DA. Gp120 also potentiated quinolinate-evoked [(3)H]NA release. Ifenprodil (0.1-0.5 microM) or CP-101,606 (0.1-10 microM) inhibited the NMDA plus glycine-evoked release of both [(3)H]catecholamines. Zinc (0.1-1 microM) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [(3)H]NA elicited by NMDA plus glycine, whereas the release of [(3)H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 microM) or H7 (10 microM) selectively prevented the effect of NMDA plus glycine on the release of [(3)H]NA. GF 109203X also blocked the release of [(3)H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.     

Reaction of oxidized dopamine with endogenous cysteine residues in the human dopamine transporter

There is evidence to suggest that dopamine (DA) oxidizes to form dopamine ortho-quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on protein cysteinyl residues. This reaction has been shown to inhibit dopamine uptake, as well as other biological processes. We have identified specific cysteine residues in the human dopamine transporter (hDAT) that are modified by this electron-deficient substrate analog. DAQ reactivity was inferred from its effects on the binding of [(3)H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (beta-CFT) to hDAT cysteine mutant constructs. One construct, X5C, had four cysteines mutated to alanine and one to phenylalanine (Cys(90)A, Cys(135)A, C306A, C319F and Cys(342)A). In membrane preparations 1 mM DAQ did not affect [(3)H]beta-CFT binding to X5C hDAT, in contrast to its effect in wild-type hDAT in which it reduced the B:(max) value by more than half. Wild-type cysteines were substituted back into X5C, one at a time, and the ability of DAQ to inhibit [(3)H]beta-CFT binding was assessed. Reactivity of DAQ with Cys(90) increased the affinity of [(3)H]beta-CFT for the transporter, whereas reactivity with Cys(135) decreased the affinity of [(3)H]beta-CFT. DAQ did not change the K:(D) for [(3)H]beta-CFT binding to wild-type. The reactivity of DAQ at Cys(342) decreased B:(max) to the same degree as wild-type. The latter result suggests that Cys(342) is the wild-type residue most responsible for DAQ-induced inhibition of [(3)H]beta-CFT binding.     

Cationic interactions at the human dopamine transporter reveal binding conformations for dopamine distinguishable from those for the cocaine analog 2 alpha-carbomethoxy-3 alpha-(4-fluorophenyl)tropane

In membrane preparations, CFT, a phenyltropane cocaine analog, and dopamine (DA) interact with the recombinant human dopamine transporter (hDAT) in Na+ -free medium. Na+ markedly increased the transporter's affinity for CFT, but had little or no effect on DA potency for inhibiting CFT binding. Raising [Na+ ] from 20 to 155 mm reduced Li+ -induced increase in DA K (i), but not CFT K (d). The presence of 155 mm Na+ enhanced the tolerance to low pH of CFT Kd but not DA Ki. Leucine substitution for tryptophan 84 (W84L) in transmembrane domain (TM) 1 or asparagine substitution for aspartate 313 (D313N) in TM 6 did not or only modestly enhance the affinity of Na+ -independent CFT binding, and retained the near normal ability of DA, Li+, K+, or H+ to inhibit this binding. However, the mutations significantly enhanced the Na+ stimulation of CFT binding as well as the Na+ antagonism against Li+ and H+ inhibition of CFT binding. In contrast, the mutations neither changed the Na+ -insensitive feature of DA Ki nor enhanced the Na+ protection of DA Ki against Li+ 's inhibitory effect, though they caused Na+ protection of DA Ki against H+ 's inhibitory action. These results are consistent with the existence of binding conformations for DA that are distinguishable from those for CFT, and with a differential association of cation interactions with DA and CFT binding. The mutations likely alter Na+ -bound state(s) of hDAT, preferentially strengthening the positive allosteric coupling between Na+ and CFT binding, and reducing the impact of Li+ or H+ on the CFT binding.     

Modulation of Dendritic Release of Dopamine by N-Methyl-D-Aspartate Receptors in Rat Substantia Nigra

A superfusion system was used to study the effects of excitatory amino acids (EAA) on release of [3H]dopamine ([3H]DA) previously taken up by rat substantia nigra (SN) slices. The EAA tested (20-250 microM), with the exception of quisqualate and kainate, markedly evoked [3H]DA release from nigral slices when Mg2+ ions were omitted from the superfusion medium. The EAA receptor agonists exhibited the following relative potency in stimulating [3H]DA release: L-glutamate (L-Glu) greater than N-methyl-D-aspartate (NMDA) greater than NM(D,L)A greater than D-Glu much greater than quisqualate = kainate. D-2-Amino-5-phosphonovalerate (100-200 microM), an antagonist for NMDA receptors, substantially reduced [3H]DA release evoked by L-Glu or NMDA. In contrast, L-Glu diethyl ester (100-200 microM) produced a lesser blocking effect on [3H]DA release evoked by the EAA. Further experiments showed that the NMDA-mediated release of [3H]DA was totally suppressed by the omission of Ca2+ or by the addition of tetrodotoxin (0.1 microM) to the superfusion medium. In addition, strychnine, an antagonist for glycine (Gly) receptors, significantly decreased NMDA (100 microM)-evoked as well as glycine (100 microM)-evoked release of [3H]DA from nigral slices. The results shown support the idea that activation of NMDA subtype receptors in SN may trigger a Ca2+-dependent release of DA from dendrites of nigro-striatal DA-containing neurons. Furthermore, a transsynaptic mechanism that may partially involve Gly-containing interneurons is proposed to account for some of the events mediating NMDA receptor activation and DA release in SN.     

Transmitter-Like Release of Endogenous 3,4-Dihydroxyphenylalanine from Rat Striatal Slices

Biphasic electrical field stimulation (0.5-5 Hz, 2 ms, 25 V, 3 min) and high K+ (10-30 mM, 5 min) released endogenous 3,4-dihydroxyphenylalanine (DOPA) from superfused rat striatal slices. Characteristics of the DOPA release were compared with those of 3,4-dihydroxyphenylethylamine (dopamine, DA). Electrical stimulation at 2 Hz evoked DOPA and DA over similar time courses. alpha-Methyl-p-tyrosine (0.2 mM) markedly reduced release of DOPA but not of DA. Maximal release (0.3 pmol) of DOPA was obtained at 2 Hz and at 15 mM K+. The impulse-evoked release of DOPA and DA was completely tetrodotoxin (0.3 microM) sensitive and Ca2+ dependent and the 15 mM K+-evoked release was also Ca2+ dependent. On L-[3,5-3H]tyrosine (1 microM) superfusion, high K+ (15 and 60 mM) released DOPA and DA together with concentration-dependent decreases in tyrosine 3-monooxygenase (EC 1.14.16.2) activity as indicated by [3H]H2O formation, followed by concentration-dependent increases after DOPA and DA release ended. These findings suggest that striatal DOPA is released by a Ca2+-dependent excitation-secretion coupling process similar to that involved in transmitter release.     

Rapid Kinetics of Potassium-Evoked Release of Acetylcholine from Rat Brain Synaptosomes: Analysis by Rapid Superfusion

The rapid kinetics of spontaneous and evoked [3H]acetylcholine efflux from synaptosomes was investigated using the technique of rapid superfusion. Synaptosomes were isolated from whole rat brain and the intraterminal pool of acetylcholine was radiolabeled by preincubation with [3H]choline. Synaptosomes were retained within the superfusion system on filter disks and superfused with Krebs-bicarbonate buffer, pH 7.4, at flow rates of 0.3-0.5 ml/s. These experimental conditions provided a mixing half-life of 119 ms and efficiency of superfusion of greater than 85%. The kinetics of tritium efflux was followed on the second and subsecond time scales by collection of serial 4.8-s and 50-ms samples for a total of 67.2 and 1.0 s, respectively. Superfusion for 48 s with isoosmotic Krebs buffer containing 10, 20, 30, 50, 75, and 100 mM potassium ion stimulated concentration-dependent tritium release. All of potassium-evoked release, but only 17% of spontaneous release, was calcium-dependent. Kinetic analysis of net (total minus spontaneous) potassium-stimulated release revealed a single calcium-dependent component of release that fit a single exponential function with a half-life of 12.7 s. Analysis of the area under the tritium efflux curves observed on the millisecond time scale revealed that 0.111, 0.550, and 0.614% net tritium release was evoked by superfusion for 750 ms with isoosmotic buffer containing 20, 50, and 100 mM KCl, respectively. Consistent with the results observed on the second time scale, a small fraction of spontaneous release and all of potassium-evoked release observed on the millisecond time scale were calcium-dependent. These data indicate that the technique of rapid superfusion can be utilized for the direct investigation of spontaneous and evoked [3H]acetylcholine release, as well as the factors that regulate this release from brain synaptosomes on the second and millisecond time scales.     

AN ADAPTATION OF THE PUSH-PULL CANNULA METHOD TO STUDY THE IN VIVO RELEASE OF [3H]DOPAMINE SYNTHESIZED FROM [3H]TYROSINE IN THE CAT CAUDATE NUCLEUS: EFFECTS OF VARIOUS PHYSICAL AND PHARMACOLOGICAL TREATMENTS

Abstract— The release of [³H]dopamine ([³H]DA) continuously synthesized from l-[3,5–³H]tyrosine from the caudate nucleus of the cat was estimated in halothane anaesthetized or‘encéphale isolé’animals. For this purpose, an improved superfusion cannula, avoiding tissue damage, was used. The best localization for the tip of the superfusion cannula was found first by determining the topographical distribution of endogenous DA within the caudate nucleus. A rostro-caudal heterogenous distribution of the transmitter was detected. In perfusion experiments, l-[3,5–³H]tyrosine was introduced continuously at a rate of 33μl/min. [³H]DA was the only catecholamine found in serial 15 min fractions as revealed by cochromatography. The spontaneous release of [³H]DA was greater in anaesthetized than in ‘encéphale isolé’ cats; it represented 150 and 100 times the blank value, respectively. Depolarization by K⁺ (30 mm) applied locally in the striatum or by electrical or mechanical stimulation of the substantia nigra caused a transitory increase in [³H]DA release. Conversely, a decrease in nerve activity induced by tetrodotoxin (5 × 10^?-⁷ m) or by electrocoagulation of the substantia nigra was associated with a decline in the amounts of [³H]DA in superfusates. A temporary reduction in [³H]DA release could also be obtained by a short-lasting cooling block of the substantia nigra. As expected, d-amphetamine (10^?-⁵ m) and benzotropine(10^?-⁷ m) added to the superfusing medium increased [³H]DA release. These pharmacological results, as well as the changes in [³H]DA release observed after various manipulations of the activity of dopaminergic neurones, confirms the validity and the high sensitivity of this approach.     

Halothane Increases Non-vesicular [<Superscript>3</Superscript>H]dopamine Release from Brain Cortical Slices

Experimental data suggest that halothane anesthesia is associated with significant changes in dopamine (DA) concentration in some brain regions but the mechanism of this effect is not well known. Rat brain cortical slices were labeled with [³H]DA to further characterize the effects of halothane on the release of this neurotransmitter from the central nervous system. Halothane induced an increase on the release of [³H]DA that was dependent on incubation time and anesthetic concentration (0.012, 0.024, 0.048, 0.072 and 0.096 mM). This effect was independent of extracellular or intracellular calcium. In addition, [³H]DA release evoked by halothane was not affected by TTX (blocker of voltage-dependent Na⁺ channels) or reserpine (a blocker of vesicular monoamine transporter). These data suggest that [³H]DA release induced by halothane is non-vesicular and would be mediated by the dopamine transporter (DAT) and norepinephrine transporter (NET). GBR 12909 and nomifensine, inhibitors of DAT, decreased the release of [³H]DA evoked by halothane. Nisoxetine, a blocker of NET, reduced the release of [³H]DA induced by halothane. In addition, GBR 12909, nisoxetine and, halothane decrease the uptake of [³H]DA into rat brain cortical slices. A decrease on halothane-induced release of [³H]DA was also observed when the brain cortical slices were incubated at low temperature and low extracellular sodium, which are known to interfere with the carrier-mediated release of the neurotransmitter. Ouabain, a Na⁺/K⁺ ATPase pump inhibitor, which induces DA release through reverse transport, decreased [³H]DA release induced by halothane. It is suggested that halothane increases [³H]DA release in brain cortical slices that is mediated by DAT and NET present in the plasma membrane.     

Analysis of VMAT2 binding after methamphetamine or MPTP treatment: disparity between homogenates and vesicle preparations

[3H]Dihydrotetrabenazine ([3H]DTBZ), a specific ligand for the vesicular monoamine transporter (VMAT2), has been used to characterize the integrity of monoaminergic nerve terminals in experimental animals and humans. The purpose of the present studies was to compare the loss of VMAT2 binding with the loss of other neurochemical markers of the dopamine (DA) nerve terminals in mice treated with neurotoxic doses of methamphetamine (METH) or MPTP. Profound decreases (> or =70%) in DA content, tyrosine hydroxylase activity, and PH]carbomethoxy-3-(4-fluorophenyl)tropane binding to the DA transporter were observed in striatal homogenates at both 1 and 6 days after exposure to the neurotoxins. It is surprising that no significant loss of [3H]DTBZ binding in the homogenates was observed at 1 day after exposure, although a significant loss (-50%) was apparent 6 days later. However, in isolated vesicle preparations, [3H]DTBZ binding and active [3H]DA uptake were markedly reduced (>70%) at 1 day. These observations indicate that vesicle function is compromised at an early time point after exposure to neurotoxic insult. Furthermore, the changes in [H]DTBZ binding in homogenates may not be a sensitive indicator of early damage to synaptic vesicles, although homogenate binding reliably identifies a loss of VMAT2 at later times.     

Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells

The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.     

Dopamine Efflux from Striatum After Chronic Nicotine: Evidence for Autoreceptor Desensitization

We examined the effect of chronic nicotine treatment on dopaminergic activity by measuring the effects of D1 and D2 dopamine (DA) receptor agonists and antagonists on tritium release from mouse striatum preloaded with [3H]DA. The radioactivity released during superfusion was separated on alumina columns and the distribution and efflux of [3H]DA and its main 3H-labeled metabolites were quantified. After preloading by incubation with [3H]DA, the electrical stimulation-evoked tritium overflow was higher in striatum prepared from nicotine-treated mice, whereas in vitro addition of nicotine caused a similar increase in tritium release from striatum of untreated and chronic nicotine-treated mice. The overflow of [3H]DA and its 3H-metabolites exhibited similar distribution patterns in [3H]DA-preloaded striatum dissected from untreated and chronic nicotine-pretreated mice, indicating that repeated injections with nicotine did not alter the metabolism of [3H]DA taken up by the tissue. (-)-Quinpirole, a selective agonist for D2 DA receptors, and apomorphine, a nonselective D1/D2 agonist, inhibited the electrical stimulation-induced tritium efflux from striatum of untreated mice, whereas (+/-)-sulpiride, a D2 DA receptor antagonist, enhanced the evoked release of tritium. These changes in tritium efflux effected by (-)-quinpirole and (+/-)-sulpiride reflected changes in [3H]DA release and not in DA metabolism, as shown by separation of the released radioactivity on alumina columns. The D1 receptor agonist (+/-)-SKF-38393 did not affect the tritium overflow, whereas the D1 receptor antagonist (+)-SCH-23390 exerted a stimulatory action but only at a high concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.     

Chronic Nicotine Administration Differentially Affects Neurotransmitter Release from Rat Striatal Slices

Abstract: The objective of these experiments was to determine whether the chronic administration of nicotine, at a dose regimen that increases the density of nicotine binding sites, alters the nicotine-induced release of [³H]dopamine ([³H]DA), [³H]norepinephrine ([³H]NE), [³H]serotonin ([³H]5-HT), or [³H]acetylcholine ([³H]ACh) from rat striatal slices. For these experiments, rats received subcutaneous injections of either saline or nicotine bitartrate [1.76 mg (3.6 µmol)/kg, dissolved in saline] twice daily for 10 days, and neurotransmitter release was measured following preloading of the tissues with [³H]DA, [³H]NE, [³H]5-HT, or [³H]choline. Chronic nicotine administration did not affect the accumulation of tritium by striatal slices, the basal release of radioactivity, or the 25 mM KCl-evoked release of neurotransmitter. Superfusion of striatal slices with 1, 10, and 100 µM nicotine increased [³H]DA release in a concentration-dependent manner, and release from slices from nicotine-injected animals was significantly (p < 0.05) greater than release from saline-injected controls; release from the former increased to 132, 191, and 172% of release from the controls following superfusion with 1, 10, and 100 µM nicotine, respectively. Similarly, [³H]5-HT release increased in a concentration-related manner following superfusion with nicotine, and release from slices from nicotine-injected rats was significantly (p < 0.05) greater than that from controls. [³H]5-HT release from slices from nicotine-injected rats evoked by superfusion with 1 and 10 µM nicotine increased to 453 and 217%, respectively, of release from slices from saline-injected animals. The nicotine-induced release of [³H]NE from striatal slices was also concentration dependent but was unaffected by chronic nicotine administration. [³H]ACh release from striatal slices could not be detected when samples were superfused with nicotine but was measurable when tissues were incubated with nicotine. The release of [³H]ACh from slices from nicotine-injected rats was significantly (p < 0.05) less than release from controls and decreased to 36, 83, and 77% of control values following incubation with 1, 10, or 100 µM nicotine, respectively. This decreased [³H]ACh release could not be attributed to methodological differences because slices from nicotine-injected rats incubated with nicotine exhibited an increased [³H]DA release, similar to results from superfusion studies. In addition, it is unlikely that the decreased release of [³H]ACh from striatal slices from nicotine-injected rats was secondary to increased DA release because [³H]ACh release from slices from hippocampus, which is not tonically inhibited by DA, also decreased significantly (p < 0.05) in response to nicotine; hippocampal slices from nicotine-injected rats incubated with 1 and 10 µM nicotine decreased to 42 and 70%, respectively, of release from slices from saline-injected animals. Results indicate that the chronic administration of nicotine increases the ability of nicotine to induce the release of [³H]DA and [³H]5-HT and decreases the ability of nicotine to evoke the release of [³H]ACh but does not alter the nicotine-induced release of [³H]NE from brain slices.     

Dopamine Inhibits Calcium-Independent γ-[3H]Aminobutyric Acid Release Induced by Kainate and High K+ in the Fish Retina

Kainic acid (KA) at micromolar concentrations stimulated the release of gamma-[3H]aminobutyric acid [( 3H]GABA) from a particulate fraction of the carp (Cyprinus carpio) retina. The KA action was dose-dependent but Ca2+-independent. A similar response was elicited by another glutamate receptor agonist, quisqualic acid, and high K+, but not by an aspartate agonist, N-methyl-D-aspartic acid. The stimulatory action of KA on the [3H]GABA release was selectively blocked by the KA blockers gamma-D-glutamylglycine and cis-2,3-piperidine dicarboxylic acid. Dopamine (DA), which is contained in DA interplexiform cells in the carp retina, inhibited the [3H]GABA release induced by KA and high K+ in a dose-dependent manner. 5-Hydroxytryptamine and two well-known GABA antagonists, bicuculline (Bic) and picrotoxin (Pic), also mimicked the DA effect on the GABA release at a comparable concentration. This inhibitory effect of DA as well as Bic and Pic on the [3H]GABA release evoked by KA was clearly antagonized by a DA blocker, haloperidol. The action of these agents (KA, DA, GABA antagonist) belonging to three different receptor categories on the GABAergic neurons (possibly external horizontal cells; H1 cells) is discussed in relation to other electrophysiological studies on the lateral spread of S-potentials between H1 cells.     

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.     

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)     

Studies of the biogenic amine transporters. 1. Dopamine reuptake blockers inhibit [3H]mazindol binding to the dopamine transporter by a competitive mechanism: Preliminary evidence for different binding domains

The present study addressed the hypothesis that the DA transporter ligand, [³H]mazindol, labels multiple sites/states associated with the dopamine (DA) transporter in striatal membranes. Incubations with [³H]mazindol proceeded for 18–24 hr at 4C in 55.2 mM sodium phosphate buffer, pH 7.4, with a protease inhibitor cocktail. In order to obtain data suitable for quantitative curve fitting, it was necessary to repurify the [³H]mazindol by HPLC before a series of experiments. Under these conditions, we observed greater than 80% specific binding. The method of binding surface analysis was used to characterize the interaction of GBR12935, BTCP, mazindol, and CFT with binding site/sites labeled by [³H]mazindol. A one site model fit the data as well as the two site model: Bmax=16911 fmol/mg protein, Kd of [³H]mazindol=75 nM, Ki of GBR12935 =8.1 nM, Ki of CFT=50 nM and Ki of BTCP=44 nM. The inhibitory mechanism (competitive or noncompetitive) of several drugs (GBR12935, CFT, BTCP, cocaine, cis-flupentixol, nomifensine, WIN35,065-2, bupropion, PCP, and benztropine) was determined. All drugs inhibited [³H]mazindol binding by a competitive mechanism. Although the ligand-selectivity of the [³H]mazindol binding site indicates that it is the uptake inhibitor recognition site of the classic DA transporter, the quantitative differences among the ligand-selectivities of different radioligands for the same site suggest that each radioligand labels different overlapping domains of the DA uptake inhibitor recognition site. It is likely that development of domain-selective drugs may further our under-standing of the DA transporter.     

Dopamine Release via Protein Kinase C Activation in the Fish Retina

Calcium-dependent phospholipid-sensitive protein kinase [protein kinase C (PKC)] was partially purified from the carp (Cyprinus carpio) retina through DE 52 ion exchange and Cellulofine gel filtration chromatography. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activated PKC in the nanomolar range. A major 38-kDa protein in the retinal supernatants (105,000 g) was phosphorylated in vitro by PKC during a short period (3 min). Other phosphoproteins also appeared during a further prolonged period (greater than 15 min). Rod-bipolar and dopamine (DA) interplexiform cells in the fish retina were immunoreactive to a monoclonal antibody to PKC (alpha/beta-subtype). The PKC antibody recognized a 78-kDa native PKC enzyme by means of an immunoblotting method. Subsequently, the effects of two kinds of PKC activators were investigated on [3H]DA release from retinal cell fractions containing DA cells that had been preloaded with [3H]DA. A phorbol ester (TPA) induced a calcium- and dose-dependent [3H]DA release during a short period (2 min), with the minimal effective dose being approximately 1 nM. Other phorbols having no tumor-promoting activity, such as 4 beta-phorbol and 4 alpha-phorbol 12,13-didecanoate, were ineffective on [3H]DA release. A synthetic diacylglycerol [1-oleoyl-2-acetylglycerol (OAG)], which is an endogenous PKC activator, was also able to induce a significant release of [3H]DA. Furthermore, TPA was found to release endogenous DA from isolated fish retina by a highly sensitive HPLC with electrochemical detection method. The OAG- or TPA-induced [3H]DA or DA release was completely blocked by inhibitors of PKC, such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)