Pharmacological characterization of dopamine transport in cultured rat astrocytes.

The effects of GBR-12909 (selective DA uptake inhibitor), zimelidine (selective 5-HT uptake inhibitor) and nisoxetine (selective NE uptake inhibitor) on the uptake of 30 nM [3H]DA into cultured rat astrocytes were examined. [3H]DA uptake was inhibited by approximately 50% by GBR-12909 or zimelidine in a concentration-dependent manner (100 nM to approximately 10 microM). Furthermore, the inhibition curves of GBR-12909 were biphasic, and uptake was completely inhibited by a high concentration of GBR-12909 (100 microM). [3H]DA uptake was also inhibited by approximately 50% by nisoxetine in a concentration-dependent manner (0.1 to approximately 100 nM), and nisoxetine was more potent than GBR-12909 or zimelidine. The inhibitory potencies were in the order nisoxetine > GBR-12909 > zimelidine. The uptake of [3H]DA under Na+-free conditions was approximately 50% of that under normal conditions. Thus, DA was taken up by both Na+-dependent and Na+-independent mechanisms. Nisoxetine (100 nM), zimelidine (100 microM) and GBR-12909 (10 microM) inhibited [3H]DA uptake into astrocytes only in the presence of Na+. On the other hand, this uptake was completely inhibited by a high concentration of GBR-12909 (100 microM) in the absence of Na+. The present data suggest that the Na+-dependent uptake of [3H]DA in cultured rat astrocytes may occur in the NE uptake system. Furthermore, astrocytes express the extraneuronal monoamine transporter (uptake2), which is an Na+-independent system, and this transporter is involved in the inactivation of centrally released DA.     

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.     

Functional expression of the norepinephrine transporter in cultured rat astrocytes

We assessed the functional expression of the norepinephrine (NE) transporter (NET) in cultured rat cortical astrocytes. Specific [3H]NE uptake increased in a time-dependent manner, and this uptake involves temperature- and Na+-sensitive mechanisms. The Na+-dependent [3H]NE uptake was saturable, and the Km for the process was 539.3 +/- 55.4 nm and the Vmax was 1.41 +/- 0.03 pmol/mg protein/min. Ouabain, a Na+-K+ ATPase inhibitor, significantly inhibited Na+-dependent [3H]NE uptake. The selective NE uptake inhibitor nisoxetine, the tricyclic antidepressants desipramine and imipramine, and the serotonin and NE reuptake inhibitor (SNRI) milnacipran very potently inhibited Na+-dependent [3H]NE uptake. On the other hand, GBR-12935 (a selective dopamine uptake inhibitor), fluvoxamine (a selective serotonin reuptake inhibitor), venlafaxine (a SNRI) and cocaine had weaker inhibitory activities. RT-PCR demonstrated that astrocytes expressed mRNA for the cloned NET protein, which was characterized as neuronal NET. Western blots indicated that anti-NET polyclonal antibody recognized a major band of 80 kDa in astrocytes. These data indicate that the neuronal NET is functionally expressed in cultured rat astrocytes. Glial cells may exert significant control of noradrenergic activity by inactivating NE that escapes neuronal re-uptake in sites distant from terminals, and are thus cellular targets for antidepressant drugs that inhibit NE uptake.     

Phosphorylation of threonine residue 276 is required for acute regulation of serotonin transporter by cyclic GMP

Cellular protein kinases, phosphatases, and other serotonin transporter (SERT) interacting proteins participate in several signaling mechanisms regulating SERT activity. The molecular mechanisms of protein kinase G (PKG)-mediated SERT regulation and the site of transporter phosphorylation were investigated. Treatment of rat midbrain synaptosomes with 8-bromo-cGMP increased SERT activity, and the increase was selectively blocked by PKG inhibitors. The V(max) value for serotonin (5-HT) transport increased following cGMP treatment. However, surface biotinylation studies showed no change in SERT surface abundance following PKG activation. (32)P metabolic labeling experiments showed increased SERT phosphorylation in the presence of cGMP that was abolished by selectively inhibiting PKG. Phosphoamino acid analysis revealed that cGMP-stimulated native SERT phosphorylation occurred only on threonine residues. When added to CHO-1 cells expressing SERT, 8-bromo-cGMP stimulated 5-HT transport and SERT phosphorylation. Mutation of SERT threonine 276 to alanine completely abolished cGMP-mediated stimulation of 5-HT transport and SERT phosphorylation. Although the T276A mutation had no significant effect on 5-HT transport or SERT protein expression, mutation to aspartate (T276D) increased the level of 5-HT uptake to that of cGMP-stimulated 5-HT uptake in wild-type SERT-expressing cells and was no longer sensitive to cGMP. These findings provide the first identification of a phosphorylation site in SERT and demonstrate that phosphorylation of Thr-276 is required for cGMP-mediated SERT regulation. They also constitute the first evidence that in the central nervous system PKG activation stimulates endogenous SERT activity by a trafficking-independent mechanism.     

Characterization of Sodium-Dependent [3H]GBR-12935 Binding in Brain: A Radioligand for Selective Labelling of the Dopamine Transport Complex

High-affinity and saturable binding sites for the diphenyl-substituted piperazine derivative [3H]GBR-12935 have been characterized in crude synaptosomal membranes prepared from rat brain. The specific binding of [3H]GBR-12935 is sodium-dependent and is unevenly distributed among various brain regions, with the highest concentration of binding sites being found in the corpus striatum and nucleus accumbens. Sodium-dependent [3H]GBR-12935 binding in all other brain areas was 10% or less of the binding found in the striatum. The affinity of [3H]GBR-12935 for binding sites in the striatum is increased in the presence of Na+ but other cations, including K+, Ca2+, or Mg2+, inhibit specific binding. There is an excellent correlation (r = 0.96, p less than 0.01) between the potencies of a series of drugs in inhibiting [3H]GBR-12935 binding to striatal membranes and their potencies in inhibiting [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine) uptake in synaptosomes. Agonists and antagonists of other neurotransmitter receptor or drug recognition sites have little or no effect on specific [3H]GBR-12935 binding to striatal membranes. In addition, prior intracerebroventricular administration of 6-hydroxydopamine results in a decrease in the number of specific [3H]GBR-12935 binding sites in the striatum. These data indicate that [3H]GBR-12935 is a selective radioligand of the presynaptic dopamine transport complex in brain.     

[3H]GBR-12935 Binding to the Dopamine Transporter Is Decreased in the Caudate Nucleus in Parkinson''s Disease

The specific binding of [3H]GBR-12935 to membranes prepared from human caudate nucleus is saturable (Bmax 1.36 +/- 0.18 pmol/mg protein), sodium dependent and of high affinity (KD 2.34 +/- 0.18 nM). Freezing of tissue from rat brain, or refrigeration followed by freezing, results in a small but significant (less than or equal to 20%) decrease in specific [3H]GBR-12935 binding when compared to the binding observed in fresh (nonfrozen) tissue, and this decrease may account, in part, for the differences in specific binding between rat and human brain membranes. Despite small differences in binding site density between fresh and frozen tissue there is a good correlation (r = 0.98; p less than 0.01) between the potencies of a series of drugs in displacing specific [3H]GBR-12935 binding to human caudate membranes and rat striatum as well as in inhibiting dopamine uptake in rat striatal synaptosomes (r = 0.96; p less than 0.01). The specific binding of [3H]GBR-12935 to membranes prepared from the caudate nuclei of patients with Parkinson's disease is decreased compared to membranes prepared from age- and sex-matched controls. These data suggest that [3H]GBR-12935 binds in a sodium-dependent fashion to the dopamine transport complex in human brain and that specific binding is decreased by a pathological degeneration of dopaminergic neurons to the caudate nucleus.     

Serotonin (5-HT) transport in human platelets is modulated by Src-catalysed Tyr-phosphorylation of the plasma membrane transporter SERT.

BACKGROUND/AIM: platelets possess tightly regulated systems for serotonin (5-HT) transport. This study analysed whether the 5-HT transport mediated by the plasma-membrane transporter SERT is regulated by its Tyr-phosphorylation. METHODS: 5-HT transport was determined by filtration techniques, while immunoblotting procedures were adopted for detecting the Tyr-phosphorylation of SERT in human platelet fractions. RESULTS: 5-HT accumulation in platelets pre-treated with reserpine, which prevents the neurotransmitter transport into the dense granules, decreased upon cellular exposure to PP2 and SU6656, two structurally unrelated inhibitors of Src-kinases. By contrast, the protein Tyr-phosphatase inhibitor pervanadate increased the 5-HT accumulation. Anti-SERT immunostaining of the platelet fractions showed a major band displaying an apparent molecular mass of 50 kappaDa, indicating that, during the analytical procedure, SERT underwent proteolysis, which was counteracted by addition of 4 M urea in the cellular disrupting medium. The Tyr-phosphorylation degree of SERT immunoprecipitated from membrane extracts decreased by platelet treatment with SU6656 or PP2, and enhanced upon pervanadate treatment. The anti-SERT immunoprecipitates displayed anti-Src immunostaining and in vitro kinase activity towards a Src-specific peptide-substrate. Platelet treatment with PP2 or SU6656 also caused a decrease in the imipramine binding to platelets. It was concluded that the Src-mediated SERT Tyr-phosphorylation regulates the 5-HT transport by affecting the neurotransmitter binding sites.     

The dopamine transporter and cytochrome P45OIID1 (debrisoquine 4-hydroxylase) in brain: resolution and identification of two distinct [3H]GBR-12935 binding proteins

Two [3H]GBR-12935 binding proteins, identified as the dopamine transporter and cytochrome P45OIID1, were solubilized in digitonin from canine striatal membranes, and were resolved following wheat germ agglutinin (WGA)-lectin column chromatography. Protein adsorbed to and specifically eluted from WGA-lectin with N-acetylglucosamine displayed saturable, high affinity (KD approximately 3 nM), and sodium-dependent binding of [3H]GBR-12935, which was inhibited in a concentration-dependent and stereoselective manner by dopamine uptake blockers and substrates with a pharmacological profile indicative of the dopamine uptake site. Protein not adsorbed to WGA-lectin also bound [3H]-GBR-12935 with high affinity (approximately 7 nM), in a sodium-independent manner, and was insensitive to classical dopamine uptake blockers and substrates such as mazindol or dopamine, corresponding to the so-called "piperazine acceptor" site seen in native membranes. [3H]GBR-12935 binding to this latter protein was, however, inhibited by various compounds with a pharmacological profile indicative of a form of cytochrome P450 designated P45OIID1 (debrisoquine/sparteine monooxygenase) with the following rank order of inhibitory potency: GBR-12909 greater than budipine greater than alpha-lobeline greater than quinidine greater than alpha flupenthixol greater than SKF-525A greater than sparteine greater than quinine. Ki values obtained for inhibition of [3H]-GBR-12935 binding to neuronal WGA passthrough fractions by these drugs correlate well with their respective Ki values for liver P45OIID1 activity. Western blotting and immunoprecipitation analysis with rabbit anti-rat P45OIID1 antibody also supported the identity of the mazindol-insensitive [3H]GBR-12935 binding site (or piperazine acceptor site) as P45OIID1. Furthermore, a [3H]GBR-12935 binding protein with pharmacological and immunological characteristics similar to those of P45OIID1 was solubilized from both bovine and human liver membranes, and GBR-12909 was found to be a potent competitive inhibitor (Ki approximately 100 nM) of sparteine monooxygenase activity in human liver microsomes. These data clearly indicate that [3H]GBR-12935 and its analogs display similar affinities for both the dopamine transporter and neuronal P45OIID1, and that this radioligand may be a useful probe of P45OIID1 activity in brain and liver. The exact molecular and functional association (if any) between these two distinct binding protein populations remains to be established; however, it is tempting to speculate that P45OIID1 is involved in the catabolism and processing of neurotransmitters subsequent to their reuptake into target cells.     

Advances in sodium-ion coupled biogenic amine transporters.

The sodium-ion coupled transporters for 5-hydroxytryptamine (5HT), noradrenaline and dopamine function to reduce extracellular levels of biogenic amines. Over the past fifteen years selective inhibitors of these transport systems have been developed including fluoxetine, citalopram, paroxetine, litoxetine (for 5HT), nisoxetine, desipramine, maprotiline (for noradrenaline) and GBR-12935 (for dopamine). Some of these inhibitors, including drugs selective for noradrenaline transport and particularly those selective for the 5HT transport system are currently widely used in the clinical management of affective disorders. Selective biogenic amine uptake inhibitors have, in addition, provided tools to undertake molecular pharmacological and biochemical studies of their respective transporters. By this means, the rat brain 5HT and dopamine transporters have been identified as polypeptides with relative molecular masses of 73,000 and 80,000, respectively, using affinity-chromatographic purification and photoaffinity-labelling techniques. Recently, the biogenic amine transporters have been cloned and a comparison of their predicted amino acid sequences reveals that these proteins share a considerable degree of similarity with notably 12-13 transmembrane spanning domains. Perspectives for future fundamental and clinical research on biogenic amine transport systems using molecular biological techniques are discussed.     

[3H] GBR-12935 Binding to Human Cerebral Cortex Is Not to Dopamine Uptake Sites

Abstract: The binding of the dopamine uptake inhibitor [³H] GBR-12935 to 16 regions of the human brain was investigated in competition experiments with increasing concentrations of GBR-12909, mazindol, and dopamine. The methodology used included a relatively high tissue concentration (8 mg/ml) and addition of 5 m M KCI in the assay buffer. GBR-12909 inhibited 80–90% of the binding in most regions, whereas dopamine only inhibited the binding in the striatum. Mazindol inhibited only part of the cortical binding at concentrations of >1 μ M , whereas the inhibition in the caudate and the putamen also contained a high-affinity component representing the dopamine uptake site. It is concluded that the [³H] GBR-12935 binding sensitive to GBR-12909 cannot be regarded as specific binding to the dopamine uptake site because the displaceable binding most likely is not related to the dopamine uptake site.     

The Pro-inflammatory Cytokine TNF-α Regulates the Activity and Expression of the Serotonin Transporter (SERT) in Astrocytes

Pro-inflammatory cytokines have been implicated in the precipitation of depression and related disorders, and the antidepressant sensitive serotonin transporter (SERT) may be a major target for immune regulation in these disorders. Here, we focus on astrocytes, a major class of immune competent cells in the brain, to examine the effects of pro-longed treatment with tumor necrosis factor-alpha (TNF-α) on SERT activity. We first established that high-affinity serotonin uptake into C6 glioma cells occurs through a SERT-dependent mechanism. Functional SERT expression is also confirmed for primary astrocytes. In both cell types, exposure to TNF-α resulted in a dose- and time-dependent increase in SERT-mediated 5-HT uptake, which was sustained for at least 48 h post-stimulation. Further analysis in primary astrocytes revealed that TNF-α enhanced the transport capacity (V_max) of SERT-specific 5-HT uptake, suggesting enhanced transporter expression, consistent with our observation of an increase in SERT mRNA levels. We confirmed that in both, primary astrocytes and C6 glioma cells, treatment with TNF-α activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Pre-treatment with the p38 MAPK inhibitor SB203580 attenuated the TNF-α mediated stimulation of 5-HT transport in both, C6 glioma and primary astrocytes. In summary, we show that SERT gene expression and activity in astrocytes is subject to regulation by TNF-α, an effect that is at least in part dependent on p38 MAPK activation.     

Inhibitory effects of cis- and trans-resveratrol on noradrenaline and 5-hydroxytryptamine uptake and on monoamine oxidase activity

This study investigated for the first time the potential effects of cis- and trans-resveratrol (c-RESV and t-RESV) on noradrenaline (NA) and 5-hydroxytryptamine (5-HT) uptake by synaptosomes from rat brain, on 5-HT uptake by human platelets, and on monoamine oxidase (MAO) isoform activity. Both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the uptake of [3H]NA and [3H]5-HT by synaptosomes from rat brain and the uptake of [3H]5-HT by human platelets. In both experimental models, t-RESV was slightly more efficient than c-RESV. Furthermore, in synaptosomes from rat brain, the RESV isomers were less selective against [3H]5-HT uptake than the reference drug fluoxetine (0.1-30 microM). On the other hand, both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the enzymatic activity of commercial (human recombinant) MAO isoform (MAO-A and MAO-B) activity, c-RESV being slightly less effective than t-RESV. In addition, both RESV isomers were slight but significantly more selective against MAO-A than against MAO-B. Since the principal groups of drugs used in the treatment of depressive disorders are NA/5-HT uptake or MAO inhibitors, under the assumption that the RESV isomers exhibit a similar behaviour in humans in vivo, our results suggest that these natural polyphenols may be of value as structural templates for the design and development of new antidepressant drugs with two important biochemical activities combined in the same chemical structure: NA/5-HT uptake and MAO inhibitory activity.     

In vivo analysis of serotonin clearance in rat hippocampus reveals that repeated administration of p-methoxyamphetamine (PMA), but not 3,4-methylenedioxymethamphetamine (MDMA), leads to long-lasting deficits in serotonin transporter function

p-Methoxyamphetamine (PMA) has been implicated in fatalities as a result of 'ecstasy' (MDMA) overdose worldwide. Like MDMA, acute effects are associated with marked changes in serotonergic neurotransmission, but the long-term effects of PMA are poorly understood. The aim of this study was to determine the effect of repeated PMA administration on in vitro measures of neurodegeneration: serotonin (5-HT) uptake, 5-HT transporter (SERT) density and 5-HT content in the hippocampus, and compare with effects on in vivo 5-HT clearance. Male rats received PMA, MDMA (4 or 15 mg/kg s.c., twice daily) or vehicle for 4 days and 2 weeks later indices of SERT function were measured. [(3)H]5-HT uptake into synaptosomes and [(3)H]cyanoimipramine binding to the SERT were significantly reduced by both PMA and MDMA treatments. 5-HT content was reduced in MDMA-, but not PMA-treatment. In contrast, clearance of locally applied 5-HT measured in vivo by chronoamperometry was only reduced in rats treated with 15 mg/kg PMA. The finding that 5-HT clearance in vivo was unaltered by MDMA treatment suggests that in vitro measures of 5-HT axonal degeneration do not necessarily predict potential compensatory mechanisms that maintain SERT function under basal conditions.     

Determination of the reversible uptake of 3H-serotonin in synaptosomes: an adequate evaluation of the contribution of nonspecific uptake

Serotonin (5-HT) uptake in synaptosomes was studied at 0 degree C (1), at 37 degrees C in the presence of 100 microM imipramine (IIa) or 100 microM zimelidine (IIb), in the absence of Na+ ions (III) in the incubation medium. A significant decrease (P less than 0.01) of the uptake rate has been found (III greater than IIa, b greater than I). Nonspecific uptake inhibition was thought to be the cause of these differences. Determination of specific uptake, using control III, has shown Na+-dependent transport of 5-HT only on the one type of carrier (Km = 174 + 24 nM). It is concluded that determination of 5-HT nonspecific uptake, using control III, has permitted a more exact evaluation of specific uptake parameters, than determination, using controls I and IIa, b.     

Differential Regulation of the Serotonin Transporter by Vesicle-Associated Membrane Protein 2 in Cells of Neuronal versus Non-Neuronal Origin

The serotonin transporter (SERT) is a key regulator of serotonergic signalling as it mediates the re-uptake of synaptic serotonin into nerve terminals, thereby terminating or modulating its signal. It is well-known that SERT regulation is a dynamic process orchestrated by a wide array of proteins and mechanisms. However, molecular details on possible coordinated regulation of SERT activity and 5-HT release are incomplete. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, interacts with SERT. This was documented in vitro, through GST pull-down assays, by co-immunoprecipitation experiments on heterologous cells and rat hippocampal synaptosomes, and with FRET analysis in live transfected HEK-293 MSR cells. The related isoforms VAMP1 and VAMP3 also physically interact with SERT. However, comparison of the three VAMP isoforms shows that only VAMP2 possesses a functionally distinct role in relation to SERT. VAMP2 influences 5-HT uptake, cell surface expression and the delivery rate of SERT to the plasma membrane differentially in HEK-293 MSR and PC12 cells. Moreover, siRNA-mediated knock-down of endogenous VAMP2 reduces 5-HT uptake in CAD cells stably expressing low levels of heterologous SERT. Deletion and mutant analysis suggest a role for the isoform specific C-terminal domain of VAMP2 in regulating SERT function. Our data identify a novel interaction between SERT and a synaptic vesicle protein and support a link between 5-HT release and re-uptake.     

Distinct Effects of Imipramine on 5-Hydroxytryptamine Uptake Mediated by the Recombinant Rat Serotonin Transporter SERT1

Abstract: Tricyclic and nontricyclic serotonin [5-hydroxytryptamine (5-HT)] uptake inhibitors are widely used for the treatment of depression. Here, we show that both the tricyclic antidepressant imipramine and the nontricyclic antidepressant citalopram competitively inhibit 5-HT transport mediated by the recombinant rat 5-HT transporter SERT1. For citalopram, the concentration producing half-maximal transport inhibition was in the same order of magnitude as its K _D value determined by equilibrium binding. In contrast, the inhibitory potency of imipramine was more than one order of magnitude lower than its K _D value. Our data are consistent with low-affinity imipramine binding occurring at or close to the substrate recognition site, which also binds citalopram. Occupation of the high-affinity imipramine binding site on SERT1 did not affect 5-HT transport but allosterically displaced citalopram from the substrate recognition site. Consequently, low concentrations of imipramine partially protected 5-HT transport from citalopram inhibition. This protection was only observed in the presence of Na⁺ because high-affinity imipramine binding is strictly sodium-dependent. Thus, depending on which of its binding sites on SERT1 is occupied, imipramine may exert distinct effects on 5-HT uptake mediated by the recombinant rat 5-HT transporter.     

Distribution of the Two Forms of Monoamine Oxidase Within Monoaminergic Neurons of the Guinea Pig Brain

The relative distribution of type A and type B monoamine oxidase (MAO) inside and outside the monoaminergic synaptosomes in preparations from hypothalamus and striatum of the guinea pig was determined by incubation of synaptosomal preparations of these regions with low concentrations of [14C]5-hydroxytryptamine (5-HT), noradrenaline, and dopamine. The deamination within the monoaminergic synaptosomes was hindered by selective amine uptake inhibitors. In the absence of these inhibitors, both intra- and extraneuronal deamination was measured. The two forms of the enzyme were differentiated with the irreversible and selective MAO-A and MAO-B inhibitors clorgyline and selegiline (l-deprenyl), respectively. [14C]5-HT was deaminated greater than 90% by MAO-A both inside and outside the 5-hydroxytryptaminergic synaptosomes prepared from the guinea pig hypothalamus. The deamination of [14C]noradrenaline within the noradrenergic synaptosomes of the hypothalamic preparation was in the ratio 75:25% for MAO-A:MAO-B; the corresponding ratio outside these synaptosomes was 45:55%. The deamination of [14C]dopamine within dopaminergic synaptosomes in the striatal preparation was 65% type A:35% type B, whereas outside these synaptosomes the ratio was 35:65%. Because the relative amounts and the distribution of the two forms of MAO in the guinea pig brain seem to be similar to those previously detected for the human brain, the MAO in the guinea pig brain may be a good model for the MAO in the human brain.     

A pharmacological comparison of [3H]GBR12935 binding to rodent striatal and kidney homogenates: binding to dopamine transporters?

Binding of [3H]GBR12935 to homogenates of mouse and rat striatum and kidney was studied. [3H]GBR12935 bound to both tissue preparations with high affinity (mouse striatum Kd = 2.4 +/- 0.4 nM, n = 4; mouse kidney Kd = 3.8 +/- 0.9 nM, n = 4), in a saturable (striatal Bmax = 1.5 +/- 0.4 pmol/mg protein; kidney Bmax = 4.9 +/- 0.5 pmol/mg protein) and reversible manner. Saturation experiments revealed the presence of a single class of high affinity binding sites in both tissues of both species. Mouse kidney appeared to possess a greater density of [3H]GBR12935 binding sites than the striatum while the reverse situation prevailed for the rat. Although two dopamine uptake inhibitors, namely GBR12909 and benztropine, displaced [3H]GBR12935 binding from striatal and kidney homogenates with a similar affinity in both tissues of these species, unlabelled mazindol, (+/-)cocaine, nomifensine and amfonelic acid were significantly (P < 0.001-0.02) more potent inhibitors of [3H]GBR12935 binding in the striatum than in the kidney. While the pharmacological profile of [3H]GBR12935 binding in the rodent striatum compared well with that of the dopamine transporter reported previously, the pharmacology in the kidney was considerably different to that in the striatum. GBR12909 (1-30 mg/kg, i.p.), a close analog of GBR12935, induced significant antidiuretic and antinatriuretic effects in spontaneously hypertensive rats. These data suggest that while [3H]GBR12935 labels the dopamine uptake sites in the brain, it does not appear to label similar sites in the kidney. The mechanism of action of GBR12909 on sodium and water excretion remains to be determined.     

Evidence that [3H]Dopamine Is Taken Up and Released from Nondopaminergic Nerve Terminals in the Rat Substantia Nigra In Vitro

Potassium chloride (25 mM) and (+)-amphetamine (100 microM) both stimulated the release of radioactivity from slices of substantia nigra preincubated with [3H]3,4-dihydroxyphenylethylamine [( 3H]dopamine). Potassium chloride (25 mM) released radioactivity from slices of both zona compacta and zona reticulata. Prior 6-hydroxydopamine (6-OHDA) lesions of one nigrostriatal pathway did not reduce the spontaneous release of radioactivity, or the potassium chloride- or amphetamine-induced release of radioactivity from slices of nigra ipsilateral to the lesion after preincubation with [3H]dopamine. The accumulation of radioactivity following incubation of nigral slices from 6-OHDA-lesioned animals with [3H]dopamine was increased when compared to uptake into slices from intact tissue. In synaptosomal preparations of striatum, nomifensine but not desipramine or fluoxetine inhibited [3H]dopamine uptake. In contrast, nomifensine, desipramine, and fluoxetine all inhibited [3H]dopamine uptake in nigral synaptosomal preparations. Following 6-OHDA lesions of one nigrostriatal pathway the uptake of [3H]dopamine into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was substantially decreased. In contrast, bilateral electrolesions of the dorsal and medial raphe nuclei reduced [3H]dopamine uptake into nigral preparations but not into striatal synaptosomes. The uptake of [3H]5-hydroxytryptamine ([3H]5-HT) into synaptosomal preparations of substantia nigra was abolished by fluoxetine and reduced by desipramine, but was unaffected by nomifensine. In contrast, fluoxetine, desipramine, and nomifensine all inhibited [3H]5-HT uptake into striatal synaptosomal preparations. Following 6-OHDA lesions of one nigro-striatal pathway the uptake of [3H]5-HT into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of Noradrenaline and 5-Hydroxytryptamine Uptake in Intact Rat Brain

A method for the measurement of 3H-monoamine [noradrenaline (NA) and 5-hydroxytryptamine (5-HT)] uptake into intact brain following intracerebroventricular injection is described. Most of the accumulated monoamine was associated with nerve terminals (synaptosomes). Radioactivity in the synaptosomal fraction was retained by 0.45-micron filters and was osmotically sensitive, features indicative of accumulation within particles rather than binding to membranes. [3H]5-HT associated with synaptosomes was reduced in animals pretreated with the neurotoxin 5,7-dihydroxytryptamine, in parallel with a reduction in endogenous 5-HT levels. Oral administration of tricyclic antidepressants including clomipramine, desipramine, and imipramine inhibited the synaptosomal accumulation of 3H-monoamines. Overall, the features of the accumulation of 3H-monoamines, after intracerebroventricular administration, displayed many of the characteristics expected of a physiological monoamine reuptake system.