Decynium-22 Research Articles

Effects of morphine on conditioned place preference and pain are independent of uptake‑2.

Morphine changes neurotransmitter release, including norepinephrine, dopamine, and serotonin. Decynium‑22 (D22) inhibits an alternative neurotransmitter removal pathway, namely uptake‑2. Uptake‑2 includes plasma membrane monoamine transporter (PMAT) and organic cation transporters that have a low affinity, but high capacity for uptake of various monoamines such as norepinephrine, dopamine, and serotonin. This study was done to assess the effect of uptake‑2 inhibition on morphine‑induced conditioned place preference (CPP) and analgesia. In this study, the effects of morphine and/or D22 on CPP were evaluated following intraperitoneal injection in mice. Afterward, changes in motor activity were evaluated by the open field test. Using the tail‑flick model, the effects of D22 and/or morphine were evaluated on the pain threshold. The results showed that 20 mg/kg of morphine induced a place preference response. D22, at the dose of 0.03 mg/kg, caused place avoidance, while at the dose of 0.3 mg/kg, it produced a notable place preference response. Co‑administration of D22 and morphine showed that morphine reversed the CPP aversion induced by D22 at the lowest dose. Motor activity did not alter. In the tail‑flick test, morphine, at the dose of 3 mg/kg but not 1 mg/kg, increased the pain threshold. D22 induced significant analgesic responses. Co‑administration of D22 and morphine caused considerable analgesic effects. The findings revealed that D22 induced both conditioned aversion and preference depending on the dose while morphine induced CPP. Both drugs produced analgesia.

Xiaoyaosan promotes neurotransmitter transmission and alleviates CUMS-induced depression by regulating the expression of Oct1 and Oct3 in astrocytes of the prefrontal cortex

Ethnopharmacological relevanceXiaoyaosan (XYS) is a traditional prescription for the treatment of liver depression and qi stagnation, and pharmacological studies have shown that XYS has great potential to reverse depression. However, anti-depression targets and the mechanism of XYS are still not entirely clear. Aim of the studyThe present study aims to explore and verify the anti-depression mechanism of XYS. Materials and methodsThe antidepressant effect of XYS was assessed in rats with depression induced by chronic unpredictable mild stimulation (CUMS). The levels of 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE) in different brain regions were measured using ELISA. The expression of organic cation transporters (Octs) were detected by western blot and immunohistochemical techniques. Then, Decynium-22 (D22), an Octs inhibitor, was injected into the prefrontal cortex (PFC) to verify the correlation between Octs and depression-like behavior. Then, the effects of XYS on the behavior, neurotransmitter concentration, and Octs expression in D22-induced rats were examined. Finally, primary astrocytes were used to verify the mechanism of XYS exerting anti-depressant activity by regulating Octs. ResultsThe result showed that XYS had a significant positive impact on the behavior of depression rats induced by CUMS. XYS also improved the secretion of 5-HT, DA, and NE in the PFC, as well as the promotion of Oct1, Oct2, and Oct3 expression in the PFC. These results suggest that XYS has the potential to alleviate depression by enhancing the secretion of neurotransmitters. This may be related to XYS regulation of Oct's expression. When the expression of Octs was inhibited in the PFC, rats exhibited behavior similar to depression, and XYS was able to reverse this behavior, indicating that Octs play a significant role in the development of depression and XYS may exert its antidepressant effects through the regulation of Octs. Furthermore, the study also found that dopamine uptake decreased after inhibiting the expression of Octs, and XYS-containing serum could reverse the downregulation of Oct1 and Oct3 and promote intracellular dopamine homeostasis in the astrocytes. Overall, XYS may exert antidepressant effects by promoting dopamine uptake to improve neurotransmitter transport by regulating the protein expression of Oct1 and Oct3 in astrocytes. ConclusionsThe antidepressant effect of XYS may be attributed to its ability to regulate the expression of Oct1 and Oct3 in astrocytes of the PFC, thereby promoting neurotransmitter transport.

Decynium-22 affects behavior in the zebrafish light/dark test

Decynium-22 (D-22) is an inhibitor of the uptake2 system of monoamine clearance, resulting in increased levels of dopamine and norepinephrine (and in some cases serotonin) in the nervous system and elsewhere. Uptake2 is mediated by low-affinity, high-capacity transporters that are inhibited by glucocorticoids, suggesting a mechanism of fast glucocorticoid-monoamine interaction in the brain and a possible target for antidepressants. D-22 dose-dependently increased anxiety-like behavior in adult zebrafish exposed to the light/dark test, monotonically increasing scototaxis (dark preference), but affecting risk assessment with an inverted-U-shaped response. These results suggest that the uptake2 system has a role in defensive behavior in zebrafish, presenting a novel mechanism by which stress and glucocorticoids could produce fast neurobehavioral adjustments in vertebrates.

Organic Cation Transporter Blocker Decynium 22 and Substrates Metformin and Berberine Enhance Social Interaction Preference in Adult Male Mice, Perhaps by Modulating Corticosterone Release

Social withdrawal in psychiatric disorders such as schizophrenia, depression and dementia resemble impaired preference for social interaction, which is a treatment‐resistant core symptom of autism. Deficient serotonin (5‐HT) signaling is strongly implicated as a contributing factor common to these psychiatric and neurodegenerative disorders. The relative lack of clinical efficacy of selective 5‐HT reuptake inhibitors (SSRIs) to improve social behavior deficits in these conditions has brought into question their therapeutic utility for this debilitating symptom. Given the limitations associated with treating sociability deficits by targeting the 5‐HT transporter (SERT), our initial goal was to characterize the effects of blocking auxiliary ‘uptake 2’ transporters of 5‐HT on social behaviors in mice. Organic cation transporters (OCTs) are among the ancillary 5‐HT transporters found in brain, and they are blocked endogenously by steroid hormones such as corticosterone. Male BTBR T+Itpr3tf/J (BTBR) mice have elevated serum corticosterone, particularly in adolescence, and they exhibit inherent deficits in preference for social interaction. In three‐chamber sociability preference tests the OCT blocker decynium‐22 (D‐22) enhanced social interactions in otherwise socially‐deficient BTBR mice, and was without adverse effect on the social behaviors of C57BL/6J (B6) mice (p < 0.05, N= 8–9). Furthermore, D‐22, and OCT substrates, berberine and metformin, all similarly attenuated the social dominance of BTBR mice in tube tests. However in the course of our studies we also found that the alkaloid antibiotic berberine and the anti‐diabetic drug metformin also had sociability enhancing properties similar to D‐22 in BTBR mice. Yet as compared to D‐22 these substrates are poor blockers of synaptosomal 5‐HT uptake. For example, a lower concentration of D‐22 is required to inhibit cortical [3H] 5‐HT uptake, with a Michaelis constant (Km) ≈ 0.5 μM, while 500x more berberine is required to equivalently inhibit it (Km ≈ 240 μM), and metformin is an even less potent 5‐HT uptake inhibitor (Km > 1000 μM, N = 3). However we and others have observed that D‐22, berberine and metformin can attenuate circulating corticosterone levels. Our findings confirm that systemic OCT blockade by D‐22 may be a promising strategy for improving social behavior, but the mechanism of action of berberine and metformin to similarly do so remain unclear. This warrants further investigation into how these compounds modulate corticosterone release, and how serum corticosterone relates to sociability impairments.Support or Funding InformationAutism Idea Award (AR110109), Department of Defense ‐ Congressionally Directed Medical Research Programs; South Texas Advanced Research Training: Undergraduate Program (R25GM097632), National Institutes of HealthD‐22 is a more potent 5‐HT uptake blocker than berberine.Figure 1

Investigating Plasma Membrane Monoamine Transporter (PMAT) as a Target for Development of Novel Antidepressant Treatments for Juveniles

Depression is a psychiatric illness that affects individuals of all ages, yet only two antidepressants are approved to treat depression in children – both belong to the selective serotonin reuptake inhibitor (SSRI) class. Moreover, children are less effectively treated by SSRIs than adults. SSRIs block reuptake of serotonin via the high‐affinity, low‐capacity serotonin transporter (SERT). The resulting increase in extracellular serotonin is thought to initiate a cascade of downstream effects, which underlie the therapeutic utility of SSRIs. However, other transporters also clear serotonin from extracellular fluid, including the low‐affinity, high‐capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT). Our lab has shown that, in adults, decynium‐22 (D22), an inhibitor of OCTs and PMAT, produces antidepressant‐like effects when SERT function is either genetically or pharmacologically impaired. However, it is unknown whether OCTs and/or PMAT may be useful targets for therapeutic interventions in juveniles. In contrast to adults, our preliminary studies show that D22 has antidepressant‐like effects in juvenile (postnatal day 21 (P21)) wild‐type mice, suggesting that OCTs and/or PMAT may be functionally upregulated in juvenile mice. Since PMAT has a higher affinity for serotonin compared to OCTs, we investigated the antidepressant‐like effects of D22 in male and female P21 PMAT wild‐type (+/+) and constitutive PMAT knockout (−/−) mice and found the antidepressant‐like effects of D22 were lost in P21 PMAT−/− mice. Using in vivo chronoamperometry, we examined serotonin clearance kinetics in CA3 region of hippocampus in P21 PMAT+/+ and PMAT−/− mice. Across serotonin concentrations ranging from 0.25 to 1.5 micromolar, we found no difference in serotonin clearance rate between genotypes. These data suggest that either a) these concentrations of serotonin are insufficient to engage PMAT, and/or b) SERT, or other OCT isoform, is upregulated in constitutive PMAT−/− mice. In either case, detection of PMAT‐dependent serotonin clearance would be obscured. Ongoing studies are investigating the effect of D22 on serotonin clearance in juveniles and adults over a range of serotonin concentrations, as well as evaluating PMAT expression in brain as a function of age and PMAT genotype. Together, these studies will expand our understanding of differences in serotonin transport mechanisms between juvenile and adult brain, and will aid in discovery of novel targets for the development of antidepressants with improved therapeutic efficacy for children suffering from depression.Support or Funding InformationThis work was supported by the National Institutes of Health R01 MH106978 to LCD

Electrophysiological Characterization of Novel Effects of the Uptake-2 Blocker Decynium-22 (D-22) on Dopaminergic Neurons in the Substantia Nigra Pars Compacta

Extracellular levels of dopamine (DA) and other monoamines in the brain depend not only on the classic transporters encoded by SLC6A gene family such as DAT, NET and SERT, but also a more recently identified group of low-affinity/high-capacity ‘Uptake-2’ transporters, mainly OCT3 and PMAT. The most frequently used pharmacological tool in functional studies of Uptake-2 is decynium-22 (D-22) known to block these transporters. However, the effectiveness of this drug in enhancing extracellular DA remains uncertain. Our aim was to test the hypothesis that D-22 increases extracellular levels of DA released from the somatodendritic region of dopaminergic neurons in the substantia nigra pars compacta (SNc) by reducing the OCT3/PMAT-dependent component of DA uptake. Extracellular DA was assessed indirectly, by evoking D2-IPSCs in SNc neurons following stimulated release of this neurotransmitter in midbrain slices obtained from mice. Recordings were conducted after partial inhibition of DAT with nomifensine, and after application of L-DOPA which increased the releasable DA pool. Contrary to our expectations, D-22 reduced, rather than increased, the amplitude of D2-IPSCs. Other effects included inhibition of GABAB-IPSCs and Ih current, and a reduction in firing frequency of nigral neurons. These results show that in addition to the previously known non-specific inhibitory action on α1 adrenoceptors, D-22 exerts additional off-target effects by inhibiting dopaminergic and GABAergic synaptic transmission in the SNc and the spontaneous (pacemaker) activity of nigral neurons. It remains to be established if these novel effects contribute to a reduction in spontaneous locomotor activity reported in previous studies after systemic drug administration.

Comparative analysis of novel decynium-22 analogs to inhibit transport by the low-affinity, high-capacity monoamine transporters, organic cation transporters 2 and 3, and plasma membrane monoamine transporter

Growing evidence supports involvement of low-affinity/high-capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) in regulating clearance of monoamines. Currently decynium-22 (D22) is the best pharmacological tool to study these transporters, however it does not readily discriminate among them, underscoring a need to develop compounds with greater selectivity for each of these transporters. We developed seven D22 analogs, and previously reported that some have lower affinity for α1-adrenoceptors than D22 and showed antidepressant-like activity in mice. Here, we extend these findings to determine the affinity of these analogs for OCT2, OCT3 and PMAT, as well as serotonin, norepinephrine and dopamine transporters (SERT, NET and DAT) using a combination of uptake competition with [3H]methyl-4-phenylpyridinium acetate in overexpressed HEK cells and [3H]citalopram, [3H]nisoxetine and [3H]WIN 35428 displacement binding in mouse hippocampal and striatal preparations. Like D22, all analogs showed greater binding affinities for OCT3 than OCT2 and PMAT. However, unlike D22, some analogs also showed modest affinity for SERT and DAT. Dual OCT3/SERT and/or OCT3/DAT actions of certain analogs may help explain their ability to produce antidepressant-like effects in mice and help account for our previous findings that D22 lacks antidepressant-like effects unless SERT function is either genetically or pharmacologically compromised. Though these analogs are not superior than D22 in discriminating among OCTs/PMAT, our findings point to development of compounds with combined ability to inhibit both low-affinity/high-capacity transporters, such as OCT3, and high-affinity/low-capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders.

Evaluation of the antidepressant therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22

Herein we describe the synthesis and evaluation of antidepressant properties of seven analogues (1–7) of the low affinity/high capacity transporter blocker decynium-22 (D-22). All analogues (1–7) were synthesized via base promoted coupling reactions between N-alkylated-2-methylquinolinium iodides or N-alkylated-4-methylquinolinium iodides and electrophilic N-alkylated-2-iodoquinolinium iodides. All final compounds were purified by re-crystallization or preparative HPLC and initial evaluation studies included; 1) screening for in vitro α1-adrenoceptor activity (a property that can lead to unwanted side-effects), 2) measuring antidepressant-like activity in a mouse tail suspension test (TST), and 3) measuring effects upon mouse locomotion. The results showed some analogues have lower affinities at α1-adrenoceptors compared to D-22 and showed antidepressant-like activity without the need for co-administration of SSRIs. Additionally, many analogues did not affect mouse locomotion to the same extent as D-22. Plans for additional evaluations of these promising analogues, including measurement of antidepressant-like activity with co-administration of selective serotonin re-uptake inhibitors (SSRIs), are outlined.

Differential Uptake Mechanisms of Fluorescent Substrates into Stem-Cell-Derived Serotonergic Neurons.

The actions of the neurotransmitters serotonin, dopamine, and norepinephrine are partly terminated by diffusion and in part by their uptake into neurons via the selective, high-affinity transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET), respectively. There is also growing evidence that all three monoamines are taken up into neurons by low-affinity, high-capacity organic cation transporters (OCT) and the plasma membrane monoamine transporter (PMAT). Pharmacological characterization of these low-affinity recombinant transporter proteins in heterologous expression systems has revealed that they are not antagonized by classical inhibitors of SERT, DAT, or NET but that decynium-22 (D22) antagonizes OCT3 and PMAT, whereas corticosterone and progesterone selectively inhibit OCT3. Here, we show that SERT, PMAT, and OCT3, but not OCT1 and OCT2, are coexpressed in murine stem cell-derived serotonergic neurons. Using selective antagonists, we provide evidence that uptake of the fluorescent substrates FFN511, ASP+, and 5-HT into stem cell-derived serotonergic neurons is mediated differentially by these transporters and also involves an as yet unknown transport mechanism.

Mechanisms Contributing to Lack of Antidepressant Efficacy in Juveniles and Adolescents

Depression is a major health problem where most patients are not effectively treated. This problem is compounded in children where only two antidepressants are clinically approved. Both are selective serotonin (5‐HT) reuptake inhibitors (SSRIs), which are often less efficacious in young populations compared to adults. We found the antidepressant‐like effects of SSRIs in juvenile mice (21 days post‐partum) was reduced relative to adult mice; however, there was no difference in expression of hippocampal 5‐HT transporter (SERT), the target protein of SSRIs, to account for the reduced SSRI efficacy. Increased extracellular 5‐HT after SSRI treatment is thought to trigger downstream events for therapeutic response. Our data suggests transporters capable of 5‐HT uptake other than SERT, such as organic cation transporters (OCTs) and the plasma membrane monoamine transporter (PMAT), may be preventing extracellular 5‐HT from climbing to therapeutically relevant levels after SSRI treatment. The binding site density of [3H]decynium‐22 (D22), an OCTs/PMAT blocker, was greater in juvenile mice than adults. Western blots revealed the greater [3H]D22 binding was likely driven by greater PMAT protein expression in juvenile mice. In our preliminary studies, D22 (0.01mg/kg) produced antidepressant‐like effects in juvenile but not adult mice. Using in vivo chronoamperometry, a technique which measures region specific 5‐HT clearance in brain, studies are underway to determine whether the antidepressant‐like effects of D22 in juvenile and adolescent mice are related to its ability to inhibit 5‐HT clearance.Support: NIH MH093320 / MH064489

Antidepressant potential of organic cation transporter blockade: the role of norepinephrine (1144.5)

Depression is a major health problem for which most patients are not effectively treated. This underscores a need to identify new targets for the development of antidepressants with improved efficacy. Our previous studies have shown that blockade of organic cation transporters (OCTs) and the plasma membrane monoamine transporter (PMAT) with decynium‐22 (D‐22) can produce antidepressant‐like effects, which are correlated with the ability of D‐22 to inhibit serotonin (5‐HT) clearance in brain, when the 5‐HT transporter (SERT) is pharmacologically or genetically compromised. In vitro studies show that OCT3 takes up norepinephrine (NE) more efficiently than 5‐HT, which raises the possibility that D‐22 might enhance the antidepressant‐like actions of drugs that block NE uptake via the NE transporter (NET). Using in vivo chronoamperometry, an electrochemical technique which allows second by second recording of extracellular biogenic amine concentrations, we show D‐22 increases time to clear NE from the extracellular space, and show D‐22 potentiates the effects of NET blockers venlafaxine (VEN) and desipramine (DMI) to increase clearance time of NE in the dentate gyrus and CA3 of the hippocampus. We next investigated if D‐22 can enhance the antidepressant‐like effects of VEN/DMI. Both VEN and DMI reduced immobility on the tail suspension test (TST), a test commonly used to assay antidepressant‐like activity of drugs in mice. Unlike the enhancing effect of D‐22 on increasing clearance time of NE, D‐22 did not enhance the antidepressant‐like effect of VEN/DMI in wild‐type mice. Ongoing studies are investigating the antidepressant‐like effect of D22 in SERT KO and heterozygous mice.Grant Funding Source: Supported by MH093320 (LD & WK), MH064489 (LD), NARSAD (LD) and UL1RR025767 (R25 START UP).

5-Hydroxytryptamine–Mediated Neurotransmission Modulates Spontaneous and Vagal-Evoked Glutamate Release in the Nucleus of the Solitary Tract Effect of Uptake Blockade

The effect of blockade of either 5-hydroxytryptamine (5-HT)/serotonin transporter (SERT) with citalopram or the organic cation transporter 3 (OCT3)/plasma membrane monoamine transporter (PMAT) with decynium-22 (D-22) on spontaneous and evoked release of 5-HT in the nucleus tractus solitarius (NTS) was investigated in rat brainstem slices treated with gabazine. 5-HT release was measured indirectly by changes in the frequency and amplitude of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) [in the presence of tetrodotoxin (TTX)] and evoked EPSCs. Blockade of 5-HT3 receptors with granisetron reduced, whereas the 5-HT3 agonist phenylbiguanide increased, the frequency of mEPSCs. 5-HT decreased mEPSC frequency at low concentrations and increased frequency at high concentrations. This inhibition was blocked by the 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY-100635), which was ineffective on its own, whereas the excitation was reversed by granisetron. The addition of citalopram or D-22 caused inhibition, which was prevented by 5-HT1A blockade. Thus, in the NTS, the spontaneous release of 5-HT is able to activate 5-HT3 receptors, but not 5-HT1A receptors, as the release in their vicinity is removed by uptake. The ineffectiveness of corticosterone suggests that the low-affinity, high-capacity transporter is PMAT, not OCT3. For evoked 5-HT release, only D-22 caused an increase in the amplitude of EPSCs, with a decrease in the paired pulse ratio, and increased the number of spontaneous EPSCs after 20-Hz stimulation. Thus, for the evoked release of 5-HT, the low-affinity, high-capacity transporter PMAT, but not 5-HT transporter (5-HTT)/SERT, is important in the regulation of changes in 5-HT extracellular concentration.

Decynium-22 Enhances SSRI-Induced Antidepressant-Like Effects in Mice: Uncovering Novel Targets to Treat Depression

Mood disorders cause much suffering and lost productivity worldwide, compounded by the fact that many patients are not effectively treated by currently available medications. The most commonly prescribed antidepressant drugs are the selective serotonin (5-HT) reuptake inhibitors (SSRIs), which act by blocking the high-affinity 5-HT transporter (SERT). The increase in extracellular 5-HT produced by SSRIs is thought to be critical to initiate downstream events needed for therapeutic effects. A potential explanation for their limited therapeutic efficacy is the recently characterized presence of low-affinity, high-capacity transporters for 5-HT in brain [i.e., organic cation transporters (OCTs) and plasma membrane monoamine transporter], which may limit the ability of SSRIs to increase extracellular 5-HT. Decynium-22 (D-22) is a blocker of these transporters, and using this compound we uncovered a significant role for OCTs in 5-HT uptake in mice genetically modified to have reduced or no SERT expression (Baganz et al., 2008). This raised the possibility that pharmacological inactivation of D-22-sensitive transporters might enhance the neurochemical and behavioral effects of SSRIs. Here we show that in wild-type mice D-22 enhances the effects of the SSRI fluvoxamine to inhibit 5-HT clearance and to produce antidepressant-like activity. This antidepressant-like activity of D-22 was attenuated in OCT3 KO mice, whereas the effect of D-22 to inhibit 5-HT clearance in the CA3 region of hippocampus persisted. Our findings point to OCT3, as well as other D-22-sensitive transporters, as novel targets for new antidepressant drugs with improved therapeutic potential.

Decynium‐22 enhances SSRI‐induced antidepressant effects in mice: Uncovering new targets to treat depression

A major problem in the treatment of depression is that many patients do not experience therapeutic benefit. The most commonly prescribed antidepressant drugs are the selective serotonin (5‐HT) reuptake inhibitors (SSRIs), which act by blocking the high‐affinity 5‐HT transporter (SERT). The increase in extracellular 5‐HT produced by SSRIs is thought to be critical to initiate downstream events needed for therapeutic effects. A potential explanation for their limited therapeutic efficacy is the recently characterized presence of low‐affinity, high‐capacity transporters for 5‐HT in brain, (e.g. organic cation transporters [OCTs]), which may limit the ability of SSRIs to increase extracellular 5‐HT. Decynium‐22 (D‐22) is an OCT blocker, and using this compound we uncovered a significant role for OCTs in 5‐HT uptake in SERT knockout mice, raising the possibility that pharmacological inactivation of OCTs might enhance the neurochemical and behavioral effects of SSRIs. Here we show that [3H]D‐22 binding sites are richly expressed in mouse hippocampus, and that D‐22 enhances the effects of the SSRI fluvoxamine to inhibit 5‐HT clearance and to produce antidepressant‐like activity. Our findings provide a mechanistic basis for poor therapeutic outcome following treatment with SSRIs, and point to D‐22‐sensitive transporters as novel targets for new antidepressant drugs with improved therapeutic potential. Supported by National Institutes of Health Grants R01 MH064489, R03 MH086708, Morrison Trust, and a NARSAD Independent Investigator Award.

The contribution of low‐affinity transport mechanisms to serotonin clearance in synaptosomes

Although many studies assert that the serotonin (5-HT) transporter (SERT) is the predominant mechanism controlling extracellular 5-HT concentrations, accumulating evidence suggests that low affinity, high capacity transport mechanisms may contribute more to 5-HT clearance than previously thought. The goal of this study was to quantify the contributions of SERT relative to other mechanisms in clearing extracellular 5-HT concentrations ranging from 50 nM to 1 μM in synaptosomes prepared from wild-type and SERT knockout mice using rotating disk electrode voltammetry. SERT inhibitors combined with decynium-22 (D-22), a blocker of several low-affinity transporters, blocked all uptake of 5-HT into synaptosomes. We found that SERT is responsible for the majority of synaptosomal uptake only at relatively low 5-HT concentrations, but comprises a diminishing proportion of 5-HT clearance when extracellular 5-HT increases above 100 nM. The effect of D-22 was similar in wild-type and SERT knockout synaptosomes. Thus, there was no evidence of upregulation of low-affinity mechanisms in knockout mice across the concentrations of 5-HT tested. These are surprising results, in light of the prevailing view that SERT is the primary uptake mechanism for extracellular 5-HT at physiological concentrations. We conclude that non-SERT mediated 5-HT uptake is substantial even at modest 5-HT concentrations. These findings, in conjunction with other studies, have important implications for understanding serotonergic disorders and may explain the variable efficacy and stability of patients' responses to antidepressants, such as the selective serotonin reuptake inhibitors.

Molecular cloning, functional characterization and genomic organization of four alternatively spliced isoforms of the human organic cation transporter 1 (hOCT1/SLC22A1).

In this study we report the cloning of four human OCT1 (hOCT1/SLC22A1) isoforms: a long form, hOCT1G/L554, and three shorter forms (hOCT1G/L506, hOCT1G483 and hOCT1G353). All four variants could be identified in the human glioma cell line SK-MG-1, whereas only two isoforms (hOCT1G/L554 and hOCT1G/L506) were found in human liver cDNA. The hOCT1G/L554 represents the full length hOCT1 since the sequence of this clone is more than 99% identical to previously cloned hOCT1 cDNAs. Elucidation of the gene structure of human OCT1 demonstrated that the other isolated isoforms are alternatively spliced variants. The hOCT1 gene consists of 7 exons and 6 introns. When stably expressed in human embryonic kidney (HEK293) cells, only the full length hOCT1 cDNA mediated decynium-22 (D22)-sensitive uptake of tritiated 1-methyl-4-phenylpyridinium ([3H]-MPP+).

Expression and functional characterization of the extraneuronal monoamine transporter in normal human astrocytes.

In this study we examined the functional expression of the extraneuronal monoamine transporter (EMT) in normal human astrocytes (NHA). RT-PCR with EMT-specific primers demonstrated the presence of EMT mRNA in NHA. The RT-PCR products were subjected to restriction-site analysis using three different enzymes (HinfI, SacI and BclI). The restriction patterns with the three enzymes were identical and were exactly as expected from the known restriction map of human EMT cDNA. DNA sequencing was performed for the RT-PCR products from NHA. Sequence analysis demonstrated that the sequences of RT-PCR products were identical to that of EMT. The extract of NHA was immunoblotted with anti-EMT polyclonal antibody raised against EMT polypeptides. Western blotting indicated that anti-EMT polyclonal antibody recognized a band of 63 kDa. Immunocytochemical staining using anti-EMT polyclonal antibody in NHA revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or Golgi membranes, showed a considerable level of immunoreactivity. We examined the time course of temperature-dependent [3H]MPP+ uptake in NHA for 60 min. Temperature-dependent [3H]MPP+ uptake increased in a time-dependent manner for the initial 45 min and almost reached a plateau level (8.70 +/- 0.59 pmol/mg protein) at 60 min. In the presence of 3 micro m decynium22 (D22) (the most potent EMT inhibitor), temperature-dependent [3H]MPP+ uptake was strongly reduced by 61% (3.39 +/- 0.76 pmol/mg protein at 60 min). D22-sensitive [3H]MPP+ uptake was saturable over a MPP+ concentration of 6.25-200 micro m. Km for this process was 78.01 +/- 7.64 micro m and Vmax was 295.4 +/- 12.8 pmol/mg protein/min. D22-sensitive [3H]MPP+ uptake was reduced when the astrocyte membrane potential was depolarized by increasing the concentration of K+ in the uptake buffer or by adding Ba2+ to the uptake buffer. These results provide evidence that the MPP+ transport activity in NHA is potential-sensitive. Moreover, D22-sensitive [3H]MPP+ uptake was independent of extracellular Na+. D22-sensitive [3H]MPP+ uptake was inhibited by D22, various organic cations, steroids and monoamine neurotransmitters. Our results showed that the EMT is functionally expressed in NHA and may also play a key role in the disposition of cationic drugs, neurosteroids, the neurotoxin MPP+ and monoamine neurotransmitters in the brain.

Molecular cloning, functional characterization and genomic organization of four alternatively spliced isoforms of the human organic cation transporter 1 (hOCT1/SLC22A1)

In this study we report the cloning of four human OCT1 (hOCT1/SLC22A1) isoforms: a long form, hOCT1G/L554, and three shorter forms (hOCT1G/L506, hOCT1G483 and hOCT1G353). All four variants could be identified in the human glioma cell line SK‐MG‐1, whereas only two isoforms (hOCT1G/L554 and hOCT1G/L506) were found in human liver cDNA. The hOCT1G/L554 represents the full length hOCT1 since the sequence of this clone is more than 99% identical to previously cloned hOCT1 cDNAs. Elucidation of the gene structure of human OCT1 demonstrated that the other isolated isoforms are alternatively spliced variants. The hOCT1 gene consists of 7 exons and 6 introns. When stably expressed in human embryonic kidney (HEK293) cells, only the full length hOCT1 cDNA mediated decynium‐22 (D22)‐sensitive uptake of tritiated 1‐methyl‐4‐phenylpyridinium ([3H]‐MPP+).