Plasma Membrane Monoamine Transporter 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.

Transporters involved in adult rat cortical astrocyte dopamine uptake: Kinetics, expression and pharmacological modulation.

Astrocytes, glial cells in the central nervous system, perform a multitude of homeostatic functions and are in constant bidirectional communication with neuronal cells, a concept named the tripartite synapse; however, their role in the dopamine homeostasis remains unexplored. The aim of this study was to clarify the pharmacological and molecular characteristics of dopamine transport in cultured cortical astrocytes of adult rats. In addition, we were interested in the expression of mRNA of dopamine transporters as well as dopamine receptors D1 and D2 and in the effect of dopaminergic drugs on the expression of these transporters and receptors. We have found that astrocytes possess both Na+-dependent and Na+-independent transporters. Uptake of radiolabelled dopamine was time-, temperature- and concentration-dependent and was inhibited by decynium-22, a plasma membrane monoamine transporter inhibitor, tricyclic antidepressants desipramine and nortriptyline, both inhibitors of the norepinephrine transporter. Results of transporter mRNA expression indicate that the main transporters involved in cortical astrocyte dopamine uptake are the norepinephrine transporter and plasma membrane monoamine transporter. Both dopamine receptor subtypes were identified in cortical astrocyte cultures. Twenty-four-hour treatment of astrocyte cultures with apomorphine, a D1/D2 agonist, induced upregulation of D1 receptor, norepinephrine transporter and plasma membrane monoamine transporter, whereas the latter was downregulated by haloperidol and L-DOPA. Astrocytes take up dopamine by multiple transporters and express dopamine receptors, which are sensitive to dopaminergic drugs. The findings of this study could open a promising area of research for the fine-tuning of existing therapeutic strategies.

Heterotypic Stressors Unmask Behavioral Influences of PMAT Deficiency in Mice.

Certain life stressors having enduring physiological and behavioral consequences, in part by eliciting dramatic signaling shifts in monoamine neurotransmitters. High monoamine levels can overwhelm selective transporters like the serotonin transporter. This is when polyspecific transporters like plasma membrane monoamine transporter (PMAT, Slc29a4) are hypothesized to contribute most to monoaminergic signaling regulation. Here, we employed two distinct counterbalanced stressors-fear conditioning and swim stress-in mice to systematically determine how reductions in PMAT function affect heterotypic stressor responsivity. We hypothesized that male heterozygotes would exhibit augmented stressor responses relative to female heterozygotes. Decreased PMAT function enhanced context fear expression, an effect unexpectedly obscured by a sham stress condition. Impaired cued fear extinction retention and enhanced context fear expression in males were conversely unmasked by a sham swim condition. Abrogated corticosterone levels in male heterozygotes that underwent swim stress after context fear conditioning did not map onto any measured behaviors. In sum, male heterozygous mouse fear behaviors proved malleable in response to preceding stressor or sham stress exposure. Combined, these data indicate that reduced male PMAT function elicits a form of stress-responsive plasticity. Future studies should assess how PMAT is differentially affected across sexes and identify downstream consequences of the stress-shifted corticosterone dynamics.

Impairment of exocytotic transmitter release by decynium-22 through an inhibition of ion channels.

Introduction: In addition to members of the family of Na+/Cl- dependent monoamine transporters, organic cation transporters (OCTs), in particular OCT3, as well as the plasma membrane monoamine transporter (PMAT) may contribute to neuronal reuptake of according neurotransmitters. As opposed to the numerous blockers of monoamine transporters, only a very limited number of specific blockers of OCT3 and PMAT are available. In fact, decynium-22 is the only blocking agent with micromolar affinities for both transport proteins, and this molecule is frequently used to establish roles of OCT3 and/or PMAT as targets for antidepressant drugs and psychostimulants, respectively. Methods/Results: To test for a function of these transporters in the sympathetic nervous system, uptake and release of [3H]1-methyl-4-phenylpyridinium (MPP+) was investigated in primary cultures of rat superior cervical ganglia. Uptake was reduced by cocaine or desipramine, blockers of the noradrenaline transporter, by about 70% and by corticosterone or β-estradiol, blockers of OCT3, by about 30%; decynium-22 achieved complete inhibition of uptake with half maximal effects at 3μM. Depolarization dependent release was enhanced by corticosterone or β-estradiol, but reduced by decynium-22. As the latter effect is unlikely to be related to actions at OCT3 and/or PMAT, electrophysiological recordings were performed to reveal that decynium-22 inhibits action potential firing and currents through voltage activated calcium channels in superior cervical ganglion neurons. Discussion: These results demonstrate that decynium-22 can impair exocytotic neurotransmitter release by interfering with several types of ion channels. Such transporter-independent effects of decynium-22 that my interfere with basic neuronal functions need to be considered when interpreting results obtained with decynium-22 as prototypic inhibitor of transmitter reuptake via OCT3 and/or PMAT.

Goldenseal-Mediated Inhibition of Intestinal Uptake Transporters Decreases Metformin Systemic Exposure in Mice.

Goldenseal is a perennial plant native to eastern North America. A recent clinical study reported goldenseal decreased metformin Cmax and area under the blood concentration versus time curve (AUC) by 27% and 23%, respectively, but half-life and renal clearance were unchanged. These observations suggested goldenseal altered processes involved in metformin absorption. The underlying mechanism(s) remain(s) unknown. One mechanism for the decreased metformin systemic exposure is inhibition by goldenseal of intestinal uptake transporters involved in metformin absorption. Goldenseal extract and three goldenseal alkaloids (berberine, (-)-β-hydrastine, hydrastinine) were tested as inhibitors of organic cation transporter (OCT) 3, plasma membrane monoamine transporter (PMAT), and thiamine transporter (THTR) 2 using human embryonic kidney 293 cells overexpressing each transporter. The goldenseal extract, normalized to berberine content, was the strongest inhibitor of each transporter (IC50: 4.9, 13.1, and 5.8 μM for OCT3, PMAT, and THTR2, respectively). A pharmacokinetic study in mice compared the effects of berberine, (-)-β-hydrastine, goldenseal extract, and imatinib (OCT inhibitor) on orally administered metformin. Goldenseal extract and imatinib significantly decreased metformin Cmax by 31% and 25%, respectively, and had no effect on half-life. Berberine and (-)-β-hydrastine had no effect on metformin pharmacokinetics, indicating neither alkaloid alone precipitated the interaction in vivo. A follow-up murine study involving intravenous metformin and oral inhibitors examined the contributions of basolateral enteric/hepatic uptake transporters to the goldenseal-metformin interaction. Goldenseal extract and imatinib had no effect on metformin AUC and half-life, suggesting lack of inhibition of basolateral enteric/hepatic uptake transporters. Results may have implications for patients taking goldenseal with drugs that are substrates for OCT3 and THTR2. SIGNIFICANCE STATEMENT: Goldenseal is used to self-treat respiratory infections and digestive disorders. We investigated potential mechanisms for the clinical pharmacokinetic interaction observed between goldenseal and metformin, specifically inhibition by goldenseal of intestinal uptake transporters (OCT3, PMAT, THTR2) involved in metformin absorption. Goldenseal extract inhibited all three transporters in vitro and decreased metformin systemic exposure in mice. These data may have broader implications for patients co-consuming goldenseal with other drugs that are substrates for these transporters.

The Plasma Membrane Monoamine Transporter is Highly Expressed in Neuroblastoma and Functions as an mIBG Transporter.

Neuroblastoma (NB) is a pediatric cancer with low survival rates in high-risk patients. 131I-mIBG has emerged as a promising therapy for high-risk NB and kills tumor cells by radiation. Consequently, 131I-mIBG tumor uptake and retention are major determinants for its therapeutic efficacy. mIBG enters NB cells through the norepinephrine transporter (NET), and accumulates in mitochondria through unknown mechanisms. Here we evaluated the expression of monoamine and organic cation transporters in high-risk NB tumors and explored their relationship with MYCN amplification and patient survival. We found that NB mainly expresses NET, the plasma membrane monoamine transporter (PMAT), and the vesicular membrane monoamine transporter 1/2 (VMAT1/2), and that the expression of these transporters is significantly reduced in MYCN-amplified tumor samples. PMAT expression is the highest and correlates with overall survival in high-risk NB patients without MYCN amplification. Immunostaining showed that PMAT resides intracellularly in NB cells and co-localizes with mitochondria. Using cells expressing PMAT, mIBG was identified as a PMAT substrate. In mitochondria isolated from NB cell lines, mIBG uptake was reduced by ∼50% by a PMAT inhibitor. Together, our data suggest that PMAT is a previously unrecognized transporter highly expressed in NB and could impact intracellular transport and therapeutic response to 131I-mIBG. SIGNIFICANCE STATEMENT: This study identified that plasma membrane monoamine transporter (PMAT) is a novel transporter highly expressed in neuroblastoma and its expression level is associated with overall survival rate in high-risk patients without MYCN amplification. PMAT is expressed intracellularly in neuroblastoma cells, transports meta-iodobenzylguanidine (mIBG) and thus could impact tumor retention and response to 131I-mIBG therapy. These findings have important clinical implications as PMAT could represent a novel molecular marker to help inform disease prognosis and predict response to 131I-mIBG therapy.

Inhibition of multiple uptake transporters in cardiomyocytes/mitochondria alleviates doxorubicin-induced cardiotoxicity

Doxorubicin (DOX) has been widely used to treat various tumors; however, DOX-induced cardiotoxicity limits its utilization. Since high accumulation of DOX in cardiomyocytes/mitochondria is the key reason, we aimed to clarify the mechanisms of DOX uptake and explore whether selectively inhibiting DOX uptake transporters would attenuate DOX accumulation and cardiotoxicity. Our results demonstrated that OCTN1/OCTN2/PMAT (organic cation/carnitine transporter 1/2 or plasma membrane monoamine transporter), especially OCTN2, played crucial roles in DOX uptake in cardiomyocytes, while OCTN2 and OCTN1 contributed to DOX transmembrane transport in mitochondria. Metformin (1–100 μM) concentration-dependently reduced DOX (5 μM for accumulation, 500 nM for cytotoxicity) concentration and toxicity in cardiomyocytes/mitochondria via inhibition of OCTN1-, OCTN2- and PMAT-mediated DOX uptake but did not affect its efflux. Furthermore, metformin (iv: 250 and 500 mg/kg or ig: 50, 100 and 200 mg/kg) could dose-dependently reduce DOX (8 mg/kg) accumulation in mouse myocardium and attenuated its cardiotoxicity. In addition, metformin (1–100 μM) did not impair DOX efficacy in breast cancer or leukemia cells. In conclusion, our study clarified the role of multiple transporters, especially OCTN2, in DOX uptake in cardiomyocytes/mitochondria; metformin alleviated DOX-induced cardiotoxicity without compromising its antitumor efficacy by selective inhibition of multiple transporters mediated DOX accumulation in myocardium/mitochondria.

Pharmacological Mechanism of the Non-hallucinogenic 5-HT2A Agonist Ariadne and Analogs.

Ariadne is a non-hallucinogenic analog in the phenylalkylamine chemical class of psychedelics that is closely related to an established synthetic hallucinogen, 2,5-dimethoxy-4-methyl-amphetamine (DOM), differing only by one methylene group in the α-position to the amine. Ariadne has been tested in humans including clinical trials at Bristol-Myers Company that indicate a lack of hallucinogenic effects and remarkable therapeutic effects, such as rapid remission of psychotic symptoms in schizophrenics, relaxation in catatonics, complete remission of symptoms in Parkinson's disease (PD), and improved cognition in geriatric subjects. Despite these provocative clinical results, the compound has been abandoned as a drug candidate and its molecular pharmacology remained unknown. Here, we report a detailed examination of the in vitro and in vivo pharmacology of Ariadne and its analogs, and propose a molecular hypothesis for the lack of hallucinogenic effects and the therapeutic potential of this compound class. We also provide a summary of previous clinical and preclinical results to contextualize the molecular signaling data. Our results show that Ariadne is a serotonin 5-HT2 receptor agonist, exhibits modest selectivity over 5-HT1 receptors, has no relevant activity at 5-HT4,5,7 and other aminergic receptors, and no substantial affinity at plasma membrane monoamine transporters. Compared to DOM, Ariadne shows lower signaling potency and efficacy in multiple signaling pathways examined (Gq, G11, and β-arrestin2) coupled to 5-HT2A receptors. We confirmed the shift in signaling for an α-propyl analog and provide a molecular docking rationale for the progressive decrease in signaling potency with the growing length of the α-substituent. Ariadne versus DOM exhibits no apparent change in the relative preference between Gq/11 activation and β-arrestin2 recruitment; instead, there is a small but consistent drop in efficacy in these signaling channels. Ariadne acts as a 5-HT2A agonist in vivo in mice and shows markedly attenuated head twitch response (HTR) in comparison to its hallucinogenic analogs, consistent with previous studies in rabbits, cats, and dogs. Hence, we propose the lower 5-HT2A receptor signaling efficacy of this compound class as an explanatory model for the lack of hallucinogenic effects of Ariadne in humans and the dramatically attenuated hallucinosis-like effects in animals (5-HT2A signaling efficacy hypothesis). In terms of reverse translation of the noted clinical therapeutic effects, we used an auxilin knockout model of Parkinson's disease where Ariadne rescued severe motor deficits in this mouse line, on par with the effects of l-DOPA, a notable finding considering Ariadne's lack of activity at dopamine receptors and transporters. Ariadne emerges as a prototype of a new drug class, non-hallucinogenic 5-HT2A agonists, with considerable therapeutic potential across psychiatric and neurological indications.

Summarizing studies using constitutive genetic deficiency to investigate behavioural influences of uptake 2 monoamine transporters.

Burgeoning literature demonstrates that monoamine transporters with high transport capacity but lower substrate affinity (i.e., uptake 2) contribute meaningfully to regulation of monoamine neurotransmitter signalling. However, studying behavioural influences of uptake 2 is hindered by an absence of selective inhibitors largely free of off-target, confounding effects. This contrasts with study of monoamine transporters with low transport capacity but high substrate affinity (i.e., uptake 1), for which there are many reasonably selective inhibitors. To circumvent this dearth of pharmacological tools for studying uptake 2, researchers have instead employed mice with constitutive genetic deficiency in three separate transporters. By studying baseline behavioural shifts, plus behavioural responses to environmental and pharmacological manipulations-the latter primarily targeting uptake 1-investigators have been creatively characterizing the behavioural, and often sex-specific, influences of uptake 2. This non-systematic mini review summarizes current uptake 2 behaviour literature, highlighting emphases on stress responsivity in organic cation transporter 2 (OCT2) work, psychostimulant responsivity in OCT3 and plasma membrane monoamine transporter (PMAT) investigations, and antidepressant responsivity in all three. Collectively, this small but growing body of work reiterates the necessity for development of selective uptake 2-inhibiting drugs, with reviewed studies suggesting that these might advance personalized treatment approaches.

Transporter-dependent uptake and metabolism of myocardial interstitial serotonin in the rat heart

To investigate the roles of the serotonin (5-HT) transporter (SERT) and plasma membrane monoamine transporter (PMAT) in 5-HT uptake and its metabolism in the heart, we monitored myocardial interstitial levels of 5-HT and 5-HIAA, a metabolite of 5-HT by monoamine oxidase (MAO), in anesthetized rats using a microdialysis technique. Fluoxetine (SERT inhibitor), decynium-22 (PMAT inhibitor), or their mixture was locally administered by reverse-microdialysis for 60 min. Subsequently, pargyline (MAO inhibitor) was co-administered. Fluoxetine rapidly increased dialysate 5-HT concentration, while decynium-22 gradually increased it. The mixture induced a larger increase in dialysate 5-HT concentration compared to fluoxetine or decynium-22 alone. Fluoxetine increased dialysate 5-HIAA concentration, and this increase was abolished by pargyline. Decynium-22 and the mixture did not change dialysate 5-HIAA concentration, which were not affected by pargyline. Both SERT and PMAT regulate myocardial interstitial 5-HT levels by its uptake; however, 5-HT uptake via PMAT leads to 5-HT metabolism by MAO.

Faster Serotonin Clearance in CA3 Region of Hippocampus and Antidepressant-like Effect of Decynium-22 in Juvenile Mice Are Putatively Linked to Increased Plasma Membrane Monoamine Transporter Function: Implications for Efficacy of Antidepressants in Juveniles.

Selective serotonin reuptake inhibitors (SSRIs) are less efficacious in treating depression in children than in adults. SSRIs block serotonin uptake via the high-affinity, low-capacity serotonin transporter. However, the low-affinity, high-capacity organic cation transporter 3 (OCT3) and plasma membrane monoamine transporter (PMAT) are emerging as important players in serotonin uptake. We hypothesized that OCT3 and/or PMAT are functionally upregulated in juveniles, thereby buffering SSRIs’ ability to enhance serotonergic neurotransmission. Unlike in adult mice, we found the OCT/PMAT blocker, decynium-22, to have standalone antidepressant-like effects in juveniles. Using in vivo high-speed chronoamperometry, we found that juveniles clear serotonin from the CA3 region of the hippocampus ~2-fold faster than adult mice. Cell density did not differ between ages, suggesting that faster serotonin clearance in juveniles is unrelated to faster diffusion through the extracellular matrix. Western blot and immunohistochemistry showed that juvenile mice have modestly greater expression of PMAT than adults, whereas OCT3 expression in the CA3 region of the hippocampus was similar between ages. Together, these data suggest that faster serotonin clearance and antidepressant-like effects of decynium-22 in juvenile mice may be due to functionally upregulated PMAT. Faster serotonin clearance via PMAT in juveniles may contribute to reduced therapeutic efficacy of SSRIs in children relative to adults.

Antioxidant and Neuroprotective Effects of Paeonol against Oxidative Stress and Altered Carrier-Mediated Transport System on NSC-34 Cell Lines.

Paeonol is a naturally occurring phenolic agent that attenuates neurotoxicity in neurodegenerative diseases. We aimed to investigate the antioxidant and protective effects of paeonol and determine its transport mechanism in wild-type (WT; NSC-34/hSOD1WT) and mutant-type (MT; NSC-34/hSOD1G93A) motor neuron-like amyotrophic lateral sclerosis (ALS) cell lines. Cytotoxicity induced by glutamate, lipopolysaccharides, and H2O2 reduced viability of cell; however, the addition of paeonol improved cell viability against neurotoxicity. The [3H]paeonol uptake was increased in the presence of H2O2 in both cell lines. Paeonol recovered ALS model cell lines by reducing mitochondrial oxidative stress induced by glutamate. The transport of paeonol was time-, concentration-, and pH-dependent in both NSC-34 cell lines. Kinetic parameters showed two transport sites with altered affinity and capacity in the MT cell line compared to the WT cell line. [3H]Paeonol uptake increased in the MT cell line transfected with organic anion transporter1 (Oat1)/Slc22a6 small interfering RNA compared to that in the control. Plasma membrane monoamine transporter (Pmat) was also involved in the uptake of paeonol by ALS model cell lines. Overall, paeonol exhibits neuroprotective activity via a carrier-mediated transport system and may be a beneficial therapy for preventing motor neuronal damage under ALS-like conditions.

Uncovering Functional Contributions of PMAT (Slc29a4) to Monoamine Clearance Using Pharmacobehavioral Tools

Plasma membrane monoamine transporter (PMAT, Slc29a4) transports monoamine neurotransmitters, including dopamine and serotonin, faster than more studied monoamine transporters, e.g., dopamine transporter (DAT), or serotonin transporter (SERT), but with ~400–600-fold less affinity. A considerable challenge in understanding PMAT’s monoamine clearance contributions is that no current drugs selectively inhibit PMAT. To advance knowledge about PMAT’s monoamine uptake role, and to circumvent this present challenge, we investigated how drugs that selectively block DAT/SERT influence behavioral readouts in PMAT wildtype, heterozygote, and knockout mice of both sexes. Drugs typically used as antidepressants (escitalopram, bupropion) were administered acutely for readouts in tail suspension and locomotor tests. Drugs with psychostimulant properties (cocaine, D-amphetamine) were administered repeatedly to assess initial locomotor responses plus psychostimulant-induced locomotor sensitization. Though we hypothesized that PMAT-deficient mice would exhibit augmented responses to antidepressant and psychostimulant drugs due to constitutively attenuated monoamine uptake, we instead observed sex-selective responses to antidepressant drugs in opposing directions, and subtle sex-specific reductions in psychostimulant-induced locomotor sensitization. These results suggest that PMAT functions differently across sexes, and support hypotheses that PMAT’s monoamine clearance contribution emerges when frontline transporters (e.g., DAT, SERT) are absent, saturated, and/or blocked. Thus, known human polymorphisms that reduce PMAT function could be worth investigating as contributors to varied antidepressant and psychostimulant responses.

Fear Processing in Mice with PMAT Deficiency

The plasma membrane monoamine transporter (PMAT, Slc29a4) is a polyspecific cation transporter that, in the brain, predominantly takes up monoamine neurotransmitters like dopamine and serotonin. PMAT function is hypothesized to emerge when other monoamine transporter function is impaired, such as under conditions of heightened monoamine neurotransmitter release in response to stressors. In humans, common genetic polymorphisms can reduce PMAT function, but these have exclusively been studied in the context of metformin treatment response under diabetic conditions. Nothing is currently known about how reduced PMAT function affects fear processing, nor any other aspects of emotional learning or memory. Our lab sought to begin examining the effects of PMAT deficiency on fear processing by using mice with constitutive reductions in PMAT function (i.e., heterozygotes) and comparing these to wildtype mice. Specifically, we evaluated both cued and contextual fear processing. Based on evidence that chronic pharmacological blockade of serotonin transporter‐mediated monoamine uptake decreased both cued and contextual fear expression, we hypothesized heterozygote mice would display lower levels of both cued and contextual fear relative to wildtypes. Surprisingly, we only observed a trend (p=0.08) specifically in male heterozygotes for attenuated cued fear expression. Otherwise, heterozygotes of both sexes exhibited cued fear acquisition, extinction retention, and renewal that was indistinguishable from wildtypes, as was their contextual fear processing. Four weeks after fear processing behavior tests concluded, mice were subjected to a brief swim stress, and blood was collected for stress hormone quantification. These data are currently being analyzed, and we hypothesize that heterotypic stressor exposure will unmask the behavioral sequelae of reduced PMAT function. Our current findings are consistent with previous reports of sex‐selective effects of PMAT deficiency on behavioral responses to stressor exposure. Continued investigation into how reduced PMAT function affects emotion processing and stress responsivity could have translational implications for people with functional PMAT polymorphisms.

Estradiol reduced 5-HT reuptake by downregulating the gene expression of Plasma Membrane Monoamine Transporter (PMAT, Slc29a4) through estrogen receptor β and the MAPK/ERK signaling pathway

Estrogen deficiency-induced female depression is closely related to 5-hydroxytriptamine (5-HT) deficiency. Estradiol (17β-estradiol, E2) regulates the monoamine transporters and acts as an antidepressant by affecting 5-HT clearance through estrogen receptors and related signaling pathways at the genomic level, although the specific mechanisms require further exploration. The brain expresses higher levels of plasma membrane monoamine transporter (PMAT, involved in 5-HT reuptake of the uptake 2 system) than other uptake transporters. In this study, we found that estrogen-deficient ovariectomized (OVX) rats had high PMAT mRNA and protein expression levels in the hippocampus and estradiol significantly reduced these levels. Furthermore, estradiol inhibited PMAT expression and reduced 5-HT reuptake in neurons and astrocytes and estradiol regulated the PMAT expression mainly by affecting estrogen receptor β (ERβ) at the genomic level in astrocytes. Further experiments showed that estradiol also regulated PMAT expression through the MAPK/ERK signaling pathway and not through the PI3K/AKT signaling pathway. In conclusion, estradiol inhibited 5-HT reuptake by regulating PMAT expression at the genomic level through ERβ and the MAPK/ERK signaling pathway, highlighting the importance of PMAT in the antidepressant effects of estradiol through 5-HT clearance reduction.

Live Tissue Imaging Reveals Distinct Transcellular Pathways for Organic Cations and Anions at the Blood-Cerebrospinal Fluid Barrier.

Formed by the choroid plexus epithelial (CPE) cells, the blood-cerebrospinal fluid barrier (BCSFB) plays an active role in removing drugs, toxins, and metabolic wastes from the brain. Several organic cation and anion transporters are expressed in the CPE cells, but how they functionally mediate transepithelial transport of organic cations and anions remain unclear. In this study, we visualized the transcellular transport of fluorescent organic cation and organic anion probes using live tissue imaging in freshly isolated mouse choroid plexuses (CPs). The cationic probe, 4-[4-(dimethylamino)phenyl]-1-methylpyridinium iodide (IDT307) was transported into CPE cells at the apical membrane and highly accumulated in mitochondria. Consistent with the lack of expression of organic cation efflux transporters, there was little efflux of IDT307 into the blood capillary space. Furthermore, IDT307 uptake and intracellular accumulation was attenuated by approximately 70% in CP tissues from mice with targeted deletion of the plasma membrane monoamine transporter (Pmat). In contrast, the anionic probe fluorescein-methotrexate (FL-MTX) was rapidly transported across the CPE cells into the capillary space with little intracellular accumulation. Rifampicin, an inhibitor of organic anion transporting polypeptides (OATPs), completely blocked FL-MTX uptake into the CPE cells whereas MK-571, a pan-inhibitor of multidrug resistance associated proteins (MRPs), abolished basolateral efflux of FL-MTX. In summary, our results suggest distinct transcellular transport pathways for organic cations and anions at the BCSFB and reveal a pivotal role of PMAT, OATP and MRP transporters in organic cation and anion transport at the blood-cerebrospinal fluid interface. SIGNIFICANCE STATEMENT: Live tissue imaging revealed that while organic cations are transported from the cerebrospinal fluid (CSF) into the choroid plexus epithelial cells by plasma membrane monoamine transporter without efflux into the blood, amphipathic anions in the CSF are efficiently transported across the BCSFB through the collaborated function of apical organic anion transporting polypeptides and basolateral multidrug resistance associated proteins. These findings contribute to a mechanistic understanding of the molecular and cellular pathways for choroid plexus clearance of solutes from the brain.

Role of Organic Cation Transporter 3 and Plasma Membrane Monoamine Transporter in the Rewarding Properties and Locomotor Sensitizing Effects of Amphetamine in Male andFemale Mice.

A lack of effective treatment and sex-based disparities in psychostimulant addiction and overdose warrant further investigation into mechanisms underlying the abuse-related effects of amphetamine-like stimulants. Uptake-2 transporters such as organic cation transporter 3 (OCT3) and plasma membrane monoamine transporter (PMAT), lesser studied potential targets for the actions of stimulant drugs, are known to play a role in monoaminergic neurotransmission. Our goal was to examine the roles of OCT3 and PMAT in mediating amphetamine (1 mg/kg)-induced conditioned place preference (CPP) and sensitization to its locomotor stimulant effects, in males and females, using pharmacological, decynium-22 (D22; 0.1 mg/kg, a blocker of OCT3 and PMAT) and genetic (constitutive OCT3 and PMAT knockout (−/−) mice) approaches. Our results show that OCT3 is necessary for the development of CPP to amphetamine in males, whereas in females, PMAT is necessary for the ability of D22 to prevent the development of CPP to amphetamine. Both OCT3 and PMAT appear to be important for development of sensitization to the locomotor stimulant effect of amphetamine in females, and PMAT in males. Taken together, these findings support an important, sex-dependent role of OCT3 and PMAT in the rewarding and locomotor stimulant effects of amphetamine.

Interaction Profiles of Central Nervous System Active Drugs at Human Organic Cation Transporters 1-3 and Human Plasma Membrane Monoamine Transporter.

Many psychoactive compounds have been shown to primarily interact with high-affinity and low-capacity solute carrier 6 (SLC6) monoamine transporters for norepinephrine (NET; norepinephrine transporter), dopamine (DAT; dopamine transporter) and serotonin (SERT; serotonin transporter). Previous studies indicate an overlap between the inhibitory capacities of substances at SLC6 and SLC22 human organic cation transporters (SLC22A1–3; hOCT1–3) and the human plasma membrane monoamine transporter (SLC29A4; hPMAT), which can be classified as high-capacity, low-affinity monoamine transporters. However, interactions between central nervous system active substances, the OCTs, and the functionally-related PMAT have largely been understudied. Herein, we report data from 17 psychoactive substances interacting with the SLC6 monoamine transporters, concerning their potential to interact with the human OCT isoforms and hPMAT by utilizing radiotracer-based in vitro uptake inhibition assays at stably expressing human embryonic kidney 293 cells (HEK293) cells. Many compounds inhibit substrate uptake by hOCT1 and hOCT2 in the low micromolar range, whereas only a few substances interact with hOCT3 and hPMAT. Interestingly, methylphenidate and ketamine selectively interact with hOCT1 or hOCT2, respectively. Additionally, 3,4-methylenedioxymethamphetamine (MDMA) is a potent inhibitor of hOCT1 and 2 and hPMAT. Enantiospecific differences of R- and S--pyrrolidinovalerophenone (R- and S--PVP) and R- and S-citalopram and the effects of aromatic substituents are explored. Our results highlight the significance of investigating drug interactions with hOCTs and hPMAT, due to their role in regulating monoamine concentrations and xenobiotic clearance.

Serotonin modulates melatonin synthesis as an autocrine neurotransmitter in the pineal gland

The pineal gland secretes melatonin principally at night. Regulated by norepinephrine released from sympathetic nerve terminals, adrenergic receptors on pinealocytes activate aralkylamine N-acetyltransferase that converts 5-hydroxytryptamine (5-HT, serotonin) to N-acetylserotonin, the precursor of melatonin. Previous studies from our group and others reveal significant constitutive secretion of 5-HT from pinealocytes. Here, using mass spectrometry, we demonstrated that the 5-HT is secreted primarily via a decynium-22-sensitive equilibrative plasma membrane monoamine transporter instead of by typical exocytotic quantal secretion. Activation of the endogenous 5-HT receptors on pinealocytes evoked an intracellular Ca2+ rise that was blocked by RS-102221, an antagonist of 5-HT2C receptors. Applied 5-HT did not evoke melatonin secretion by itself, but it did potentiate melatonin secretion evoked by submaximal norepinephrine. In addition, RS-102221 reduced the norepinephrine-induced melatonin secretion in strips of pineal gland, even when no exogenous 5-HT was added, suggesting that the 5-HT that is constitutively released from pinealocytes accumulates enough in the tissue to act as an autocrine feedback signal sensitizing melatonin release.

Differential Serotonin Uptake Mechanisms at the Human Maternal-Fetal Interface.

Serotonin (5-HT) plays an extensive role during pregnancy in regulating both the placental physiology and embryonic/fetal development. The uptake of 5-HT into cells is central to the control of local concentrations of 5-HT near its molecular targets. Here, we investigated the mechanisms of 5-HT uptake into human primary placental cells and cord blood platelets, all isolated immediately after birth. Trophoblasts and cord blood platelets showed 5-HT uptake with similar Michaelis constant (Km) values (~0.6 μM), typical of the high-affinity serotonin transporter (SERT). The uptake of 5-HT into trophoblasts was efficiently inhibited by various SERT-targeting drugs. In contrast, the uptake of 5-HT into feto-placental endothelial cells was not inhibited by a SERT blocker and showed a Km value (~782 μM) in the low-affinity range. Consistent with this, SERT mRNAs were abundant in term trophoblasts but sparse in feto-placental endothelial cells, whereas the opposite was found for the low-affinity plasma membrane monoamine transporter (PMAT) mRNAs. Organic cation transporter (OCT) 1, 2, and 3 mRNAs were absent or sparse in both cell types. In summary, the results demonstrate, for the first time, the presence of functional 5-HT uptake systems in feto-placental endothelial cells and fetal platelets, cells that are in direct contact with fetal blood plasma. The data also highlight the sensitivity to various psychotropic drugs of 5-HT transport into trophoblasts facing the maternal blood. The multiple, high-, and low-affinity systems present for the cellular uptake of 5-HT underscore the importance of 5-HT homeostasis at the maternal–fetal interface.