DAT Cells Research Articles

Measuring inhibition of monoamine reuptake transporters by new psychoactive substances (NPS) in real-time using a high-throughput, fluorescence-based assay

The prevalence and use of new psychoactive substances (NPS) is increasing and currently over 600 NPS exist. Many illicit drugs and NPS increase brain monoamine levels by inhibition and/or reversal of monoamine reuptake transporters (DAT, NET and SERT). This is often investigated using labor-intensive, radiometric endpoint measurements.We investigated the applicability of a novel and innovative assay that is based on a fluorescent monoamine mimicking substrate. DAT, NET or SERT-expressing human embryonic kidney (HEK293) cells were exposed to common drugs (cocaine, dl-amphetamine or MDMA), NPS (4-fluoroamphetamine, PMMA, α-PVP, 5-APB, 2C-B, 25B-NBOMe, 25I-NBOMe or methoxetamine) or the antidepressant fluoxetine.We demonstrate that this fluorescent microplate reader-based assay detects inhibition of different transporters by various drugs and discriminates between drugs. Most IC50 values were in line with previous results from radiometric assays and within estimated human brain concentrations. However, phenethylamines showed higher IC50 values on hSERT, possibly due to experimental differences.Compared to radiometric assays, this high-throughput fluorescent assay is uncomplicated, can measure at physiological conditions, requires no specific facilities and allows for kinetic measurements, enabling detection of transient effects. This assay is therefore a good alternative for radiometric assays to investigate effects of illicit drugs and NPS on monoamine reuptake transporters.

Adolescent Atomoxetine Treatment in a Rodent Model of ADHD: Effects on Cocaine Self-Administration and Dopamine Transporters in Frontostriatal Regions

Cocaine abuse and attention deficit/hyperactivity disorder (ADHD) are often comorbid. Preclinical research indicates that medial prefrontal (mPFC) and orbitofrontal (OFC) cortices are important neural substrates for both disorders. Using the spontaneously hypertensive rat (SHR) model of ADHD, we reported that adolescent treatment with the stimulant methylphenidate, a dopamine (DAT) and norepinephrine (NET) transporter inhibitor, enhanced cocaine self-administration during adulthood, and was associated with increased DAT function in mPFC. This study investigates the effects of atomoxetine ((R)-N-methyl-γ-(2-methylphenoxy)-benzenepropanamine hydrochloride) treatment, a selective NET inhibitor, during adolescence on cocaine self-administration and on DAT function and cell-surface expression in mPFC and OFC during adulthood. SHR acquired cocaine self-administration faster than Wistar-Kyoto and Wistar. Across cocaine doses, SHR earned more cocaine infusions and had higher progressive-ratio breakpoints than Wistar-Kyoto and Wistar, demonstrating that the SHR phenotype models comorbid ADHD and cocaine abuse. Prior atomoxetine treatment did not augment cocaine self-administration in SHR, but acquisition was enhanced in Wistar-Kyoto. No strain differences were found for DAT kinetic parameters or cellular localization in the vehicle controls. Atomoxetine did not alter DAT kinetic parameters or localization in SHR mPFC. Rather, atomoxetine decreased V(max) and DAT cell surface expression in SHR OFC, indicating that inhibition of NET by atomoxetine treatment during adolescence indirectly reduced DAT function and trafficking to the cell surface in OFC, specifically in the ADHD model. Thus, atomoxetine, unlike methylphenidate, does not enhance vulnerability to cocaine abuse in SHR and may represent an important alternative for teens with ADHD when drug addiction is a concern.

Differential expression of neuronal dopamine and serotonin transporters DAT and SERT in megakaryocytes and platelets generated from human MEG-01 megakaryoblasts

In the central nervous system, serotonergic and dopaminergic signaling is terminated by the activity of specialized transporter proteins for serotonin (SERT) and dopamine (DAT). These transporter proteins are found both on the cell surface and in intracellular transport vesicles. Trafficking between these subcellular domains regulates the efficiency of removal of extracellular neurotransmitters and hence the efficacy of neuronal signaling. Therefore, it is of high interest to gain more insight into the regulatory mechanisms of the human DAT and SERT cell surface expression in their natural surroundings, i.e., in human cells. Because it is not possible to cultivate human neuronal cells expressing these transporter proteins, there is a need to find other human cells expressing these neuronal proteins. Here, we have investigated the expression of human SERT and DAT on developing megakaryocytes and platelet-like particles derived from the megakaryocyte progenitor cell line MEG-01 upon differentiation by valproic acid (VPA) and all-trans retinoic acid (ATRA). Our results show that MEG-01 cells express SERT and DAT and that VPA and ATRA induce a significant increase of transporter expression on developing megakaryocytes and platelets. As compared to ATRA, VPA more efficiently induced SERT expression but not DAT expression. Comparable to naïve platelets and neurons, SERT was localized to both the cell surface and intracellular compartments. Hence, VPA and ATRA-treated MEG-01 cells provide a model well-suited to studying neuronal monoamine transporter expression, not only during transcription and translation but also with respect to protein trafficking to and from the cell surface.

207 Causes and consequences of glycolysis and acid pH in tumors

The prevalence and use of new psychoactive substances (NPS) is increasing and currently over 600 NPS exist. Many illicit drugs and NPS increase brain monoamine levels by inhibition and/or reversal of monoamine reuptake transporters (DAT, NET and SERT). This is often investigated using labor-intensive, radiometric endpoint measurements.We investigated the applicability of a novel and innovative assay that is based on a fluorescent monoamine mimicking substrate. DAT, NET or SERT-expressing human embryonic kidney (HEK293) cells were exposed to common drugs (cocaine, dl-amphetamine or MDMA), NPS (4-fluoroamphetamine, PMMA, α-PVP, 5-APB, 2C-B, 25B-NBOMe, 25I-NBOMe or methoxetamine) or the antidepressant fluoxetine.We demonstrate that this fluorescent microplate reader-based assay detects inhibition of different transporters by various drugs and discriminates between drugs. Most IC50 values were in line with previous results from radiometric assays and within estimated human brain concentrations. However, phenethylamines showed higher IC50 values on hSERT, possibly due to experimental differences.Compared to radiometric assays, this high-throughput fluorescent assay is uncomplicated, can measure at physiological conditions, requires no specific facilities and allows for kinetic measurements, enabling detection of transient effects. This assay is therefore a good alternative for radiometric assays to investigate effects of illicit drugs and NPS on monoamine reuptake transporters.

Involvement of laminin and integrins in adhesion and migration of junctional epithelium cells

The junctional epithelium attaches to the enamel surface with hemidesmosomes (of which laminin-5 and integrin-alpha(6)beta(4) are the main components) in the internal basal lamina. Laminin-5 is also involved in cell motility with integrin-alpha(3)beta(1), although their functions have not yet been clarified.The purpose of this study was to determine the functions of those adhesive components between the tooth and the junctional epithelium during cell migration.Because an idea has been proposed that directly attached to tooth cells (DAT cells) may not contribute to cell migration, 5-bromo-2-deoxyuridine staining was performed to confirm cell migration. We investigated laminin-gamma(2) (contained only in laminin-5), integrin-beta(4) (involved in cell-extracellular matrix contact) and integrin-alpha(3) (inducing cell migration) in the junctional epithelium, oral gingival epithelium and gingival sulcus epithelium of 6-wk-old ICR mice using laser microdissection, quantitative real-time reverse transcription-polymerase chain reaction, immunofluorescence and 5-bromo-2-deoxyuridine staining. Laminin and integrins were clearly immuno-localized in the basal lamina of all epithelium. Quantitative analysis of laminin and integrin mRNAs by laser microdissection showed that they were more highly expressed in DAT cells than in basal cells in the oral gingival epithelium. In particular, a 12-fold higher expression of laminin-5 was observed in the junctional epithelium compared with the oral gingival epithelium. 5-Bromo-2-deoxyuridine staining showed rapid coronal migration of DAT cells. These results suggest that the abundant expression of laminin-5 and integrin-alpha(6)beta(4) is involved in the attachment of DAT cells to teeth by hemidesmosomes. Abundant expression of laminin-5 and integrin-alpha(3)beta(1) might assist in DAT cell migration, confirmed by 5-bromo-2-deoxyuridine staining during the turnover of junctional epithelium.

Anomalous Dopamine Release Associated with a Human Dopamine Transporter Coding Variant

Dopamine (DA) signaling at synapses is tightly coordinated through opposing mechanisms of vesicular fusion-mediated DA release and transporter-mediated DA clearance. Altered brain DA signaling is suspected to underlie multiple brain disorders, including schizophrenia, Parkinson's disease, bipolar disorder, and attention-deficit hyperactivity disorder (ADHD). We identified a pedigree containing two male children diagnosed with ADHD who share a rare human DA transporter (DAT; SLC6A3) coding variant, Ala559Val. Among >1000 control and affected subjects, the Val559 variant has only been isolated once previously, in a female subject with bipolar disorder. Although hDAT Ala559Val supports normal DAT protein and cell surface expression, as well as normal DA uptake, the variant exhibits anomalous DA efflux from DA-loaded cells. We also demonstrate that hDAT Ala599Val exhibits increased sensitivity to intracellular Na(+), but not intracellular DA, and displays exaggerated DA efflux at depolarized potentials. Remarkably, the two most common ADHD medications, amphetamine and methylphenidate, both block hDAT Ala559Val-mediated DA efflux, whereas these drugs have opposite actions at wild-type hDAT. Our findings reveal that DA efflux, typically associated with amphetamine-like psychostimulants, can be produced through a heritable change in hDAT structure. Because multiple gene products are known to coordinate to support amphetamine-mediated DA efflux, the properties of hDAT Ala559Val may have broader significance in identifying a new mechanism through which DA signaling disorders arise. Additionally, they suggest that block of inappropriate neurotransmitter efflux may be an unsuspected mechanism supporting the therapeutic actions of existing transporter-directed medications.

P23 Recurrent Hyperhaemolysis

Hyperhaemolysis is a rare complication of blood transfusion in patients with SCD. An 8 year‐old boy with SCD was on a regular transfusion programme because of right frontal lobe ischaemia. He was admitted to hospital 5 days after receiving transfusion with Hb 6.5 g dL−1, reticulocyte count 330 × 109 L−1 and total bilirubin 84 μmol L−1. Haemoglobinuria was demonstrated. He was treated with oral hydration, ibuprofen and morphine for a vaso‐occlusive crisis. His Hb continued to drop to 4.1 g dL−1, reticulocyte count to 170 × 109 L−1.The DAT was negative and no red cell alloantibodies were identified. A diagnosis of hyperhaemolysis syndrome was made and he was commenced on IVIg 1 g kg−1 for 2 days and prednisolone 1 mg kg−1 for 1 week. His Hb gradually improved over a week to 6.2 g dL−1 and he did not receive transfusions. Six months later he was re‐started on a transfusion programme because of nocturnal hypoxaemia. A week after the first transfusion he was admitted with a history of lower limb and abdominal pain.Hb 7.8 g dL−1, reticulocyte count 297 × 109 L−1 and total bilirubin 106 μmol L−1 and LDH 6000 IU L−1. The Hb dropped to 3.2 g dL−1, reticulocyte count further dropped to 168 × 109 L−1 with evidence of haemoglobinuria. He developed cardiorespiratory failure requiring transfer to intensive care unit. He was given IVIg 0.5 g kg−1 for four days and three doses of methylprednisolone 4 mg kg−1 and received two RBCs units. DAT and red cell alloantibody screen were negative. He also received Erythropoietin 500 units subcutaneously three times a week. He improved over 10 days and at discharge had Hb 7.1 g dL−1, reticulocyte count 220, LDH 2033 ΙU L−1 and total bilirrubin 20 μmol L−1. He is currently on hydroxycarbamide with a stable Hb and no evidence of red cell alloantibodies. We report a case of recurrent hyperhaemolysis which is extremely rare as there were only two cases reported in the literature. The first was an adult SCD patient who had a recurrent hyperhaemolysis 27 months after the initial episode. The second was a paediatric SCD patient who had three separate episodes of hyperhaemolysis. Second and third episodes occured when additional transfusion were given 6 and 12 months after the original episode. Hyperhaemolysis may recur. It is not possible to predict which patient may recur after receiving blood. In life threatening recurrent hyperhaemolysis, use of IVIg and steroids may be life saving.

Lactoferrin impedes epithelial cell adhesion in vitro.

In the process of host defence against microbial challenge, neutrophils release granule contents with the potential side effect of damaging structural tissues. In the junctional epithelium such damage may contribute to the degeneration and renewal of the epithelial cells attached directly to the tooth (DAT cells), and subsequently to periodontal pocket formation. This study reports on lactoferrin, one of the substances released by neutrophils, and its effects on epithelial cell adhesion, growth, DNA synthesis and spreading of cell colonies at concentrations recorded in the crevicular fluid. We show that, in opposition to what has been reported on bacterial cells, lactoferrin has no effect on the DNA synthesis of attached epithelial cells in model systems attempting to simulate the DAT cells in vivo. However, both iron-saturated and unsaturated lactoferrin hampered cell adhesion, growth and spreading of cell colonies in a dose-dependent manner. These findings suggest that lactoferrin does not affect epithelial cell proliferation but it may have a role in delaying the repair of the DAT cell population during inflammation by interfering with cell adhesion.

Induction by low Na+ or Cl- of cocaine sensitive carrier-mediated efflux of amines from cells transfected with the cloned human catecholamine transporters.

1. COS-7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [3H]-dopamine or [3H]-noradrenaline and superfused with buffers of different ionic composition. 2. In DAT cells lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux. Cocaine (10 microM) or mazindol (0.3 microM) blocked the efflux at low Na+, but not at 0 Na+. Lowering the Cl- concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 microM) was without effect in all the conditions tested. 3. In NAT cells, lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na+ than at 0 mM Na+. Efflux induced by 0, 5 or 10 mM Cl- was completely blocked by all three uptake inhibitors. 4. In cross-loading experiments, 5 mM Na(+)- or 0 Cl(-)-induced efflux was much lower from [3H]-noradrenaline-loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [3H]-dopamine-loaded NAT cells elicited by 5 mM Na+ or 0 Cl- was blocked by mazindol, as well as by desipramine. 5. Thus cloned catecholamine transporters display carrier-mediated efflux of amines if challenged by lowering the extracellular Na+ or Cl-, whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors.

Bacterial metabolites sodium butyrate and propionate inhibit epithelial cell growth in vitro.

The structural and functional barrier preventing the free advancement of microbial plaque subgingivally along the tooth surface is formed by the junctional epithelial (JE) cells directly attached to the tooth (DAT cells). The mechanism leading to degeneration of the DAT cells is not known. In the present study we examined the possible role of short chain fatty acids (SCFAs) on epithelial cells by making use of 2 epithelial cell cultures (HaCaT and ERM) and an explant culture model of human JE. The SCFAs butyrate and propionate were used in concentrations found in human plaque and gingival crevicular fluid (0.25-16.0 mM). The SCFAs had no effect on primary cell adhesion nor on the epithelial attachment apparatus (EAA). By contrast, even 0.25 mM of butyrate significantly retarded epithelial cell growth. Similar effects with propionate were first observed at concentrations higher than 1.0 mM. The retardation of epithelial cell growth was found to be due to inhibition of cell division. Furthermore, after butyrate treatment dense accumulations of intermediate filaments and cytoplasmic vacuolization were characteristically seen in cells adjacent to cells of normal appearance. This suggests that some cells of the growing epithelial cell population are more sensitive to the SCFAs than others, and agrees with previous reports on the DAT cells of periodontally-involved teeth in vivo. The results suggest that SCFAs are microbial factors that play a role in the initiation and progression of periodontal pocket formation by impairing epithelial cell function rather than having a direct effect on the EAA.

Experimental Allergic Encephalomyelitis and Vaccination-Induced Resistance in DA Rats

In this report, we show that DA rats (RT1ahaplotype) immunized with myelin basic protein (MBP)-CFA develop and recover from an ascending paralysis, with the course and severity of clinical disease similar to the kinetics observed with MBP-CFA-immunized Lewis rats. Experimental allergic encephalomyelitis (EAE) can be adoptively transferred with MBP-stimulated immune spleen cells, with onset of paralysis 4 days following transfer and complete recovery 3–4 days later. To determine if the vaccination-induced resistance response could develop in the DA rat strain, which has previously been shown to occur only in the Lewis rat, we selected a MBP-specific T-cell line by standard methods from DA rats immunized previously with MBP-CFA. The DA T-cell line was encephalitogenic, and DA recipients developed and recovered from T-cell line-mediated paralytic disease. Following recovery from T-cell line-mediated disease, DA recipients were resistant to subsequent disease induction following MBP-CFA challenge, a response consistent with T-cell vaccination, as observed previously in Lewis rats. Analysis of the proliferative response of the DA T-cell line showed that the encephalitogenic fragment was within the 40–67 region of MBP, with no response to the 85–97 fragment. The 85–97 fragment, which is a minor encephalitogenic determinant for the Lewis strain, also appears to be a minor encephalitogenic epitope for DA rats. These results show that the vaccination-induced resistance response occurs in the DA rat strain and that this phenomenon is not unique to the Lewis rat model.

Characterization of the Human Junctional Epithelial Cells Directly Attached to the Tooth (DAT Cells)in Periodontal Disease

This study examined the directly attached cells (DAT cells) of the human junctional epithelium from teeth extracted for advanced periodontal disease. The monolayer nature of the DAT cells remaining on the tooth surface after extraction offers a unique opportunity to study their morphology and activity in situ. We collected DAT cells and examined them by transmission electron microscopy and by autoradiography of cells labeled with 3H-thymidine. Our morphological results suggest that degenerative changes associated with pocket formation affect individual cells rather than regions of the DAT cell population at one time. The epithelial attachment apparatus (EAA) appeared to be the most resistant structure persisting on the tooth surface during the degeneration of the individual DAT cells. The new technique developed for two-dimensional observation of the sheet of DAT cells labeled in situ on the tooth surface in culture showed that the attached cells, even in periodontal disease, exhibit proliferative ability, suggesting a regenerative role for the DAT cells.

Proliferative potential of the attached cells of human junctional epithelium

Samples of the attached cells of junctional epithelium (DAT cells) were obtained during extractions of eight erupted bicuspids from young healthy subjects. Each extracted tooth, with its layer of living DAT cells, was incubated for three hours in minimum essential medium (MEM) containing 5 microCi/ml of 3H-thymidine, and prepared for autoradiography. An average of 3 labeled cells per 1 mm of the epithelial attachment was found along the vertical dimension of the tooth. The attachment of an individual cell to the tooth surface was found to be a prerequisite for the cell's DNA synthesis suggesting that the tooth has a nonspecific epigenetic role in defining the functional phenotype of the DAT cells. Even the most coronal DAT cells located about 200 microns apical from the plaque border, were found to synthesize DNA. The finding suggests that mechanisms which regulate the multiplication of DAT cells are involved in the preservation of the integrity of the dentogingival junction.

Synthesis of type VIII collagen by epithelial cells of human gingiva

The composition of the extracellular matrix produced by epithelial cells in contact with metabolically inert substrata was studied with antibodies specific for type VIII collagen. Type VIII collagen was present in the cell layer of cultured gingival epithelial cells, and at the epithelium-substratum interface of an explant culture model for junctional epithelium. Samples of the epithelial attachment apparatus (EAA) in vivo were collected by removing the junctional cells directly attached to the tooth (DAT cells) and the associated EAA matrix. The amount of material collected was sufficient for biochemical analysis of both intra- and extracellular components of the junctional cell-EAA complex. Immunological examination of the samples revealed that type VIII collagen was associated with epithelial cells forming the EAA in vivo. We suggest that this collagen type functions in the extracellular space as an attachment-promoting factor for gingival epithelial cells at the tooth surface.

Changes in cell phenotype during regeneration of junctional epithelium of human gingiva in vitro

The relationship between cell attachment and the phenotype of the attached oral epithelial cells was studied by comparing junctional epithelium (JE) with a culture model for JE in which epithelial cells form an equivalent organization of tissues. Gingival explants were cultured on either a high or a low protein-binding membrane. The cut edge of epithelium and connective tissue was placed on the membrane; epithelial cells migrated to form a sheet of tissue between the explant and the membrane substratum. Cells which grew in contact with the high protein-binding membrane attached to the substratum and assumed a cuboidal shape. With time in culture these cells showed a decrease in reactivity with antibodies to psi-3 antigen (an antigen associated with epithelial migration) and an increase in reactivity with antibodies to cytokeratin 19 (a marker for JE). Cells grown on the low protein-binding membrane did not exhibit changes in shape or antigens. Because similar features were found in the JE in vivo, it was concluded that the junctional cells which are directly attached to the tooth (DAT cells) have a nonmigratory phenotype that develops as a response to the tooth surface. Because the cells are in contact with a metabolically inert material the changes appear to be largely self-induced. The culture method allows studies on putative inducer molecules and on mechanisms which may control the phenotype of epithelial cells at the dentogingival interface.