Decreased anxiety in mice lacking the organic cation transporter 3.

The organic cation transporter 3 (OCT3; synonymous: extraneuronal monoamine transporter, EMT, Slc22a3) encodes an isoform of the organic cation transporters and is expressed widely across the whole brain. OCTs are a family of high-capacity, bidirectional, multispecific transporters of organic cations. These also include serotonin, dopamine and norepinephrine making OCTs attractive candidates for a variety of neuropsychiatric disorders including anxiety disorders. OCT3 has been implicated in termination of monoaminergic signalling in the central nervous system. Interestingly, OCT3 mRNA is however also significantly up-regulated in the hippocampus of serotonin transporter knockout mice where it might serve as an alternative reuptake mechanism for serotonin. The examination of the behavioural phenotype of OCT3 knockout mice thus is paramount to assess the role of OCT3. We have therefore subjected mice lacking the OCT3 gene to a comprehensive behavioural test battery. While cognitive functioning in the Morris water maze test and aggression levels measured with the resident-intruder paradigm were in the same range as the respective control animals, OCT3 knockout animals showed a tendency of increased activity and were significantly less anxious in the elevated plus-maze test and the open field test as compared to their respective wild-type controls arguing for a role of OCT3 in the regulation of fear and anxiety, probably by modulating the serotonergic tone in limbic circuitries.

Impact of P-glycoprotein on clopidogrel absorption.

OBJECTIVE: The antiplatelet activity of clopidogrel is characterized by considerable interindividual differences. Variable intestinal absorption is suggested to contribute to the inconsistencies in response. We tested the hypothesis that the intestinal efflux transporter P-glycoprotein (P-gp) limits the oral bioavailability of clopidogrel and that variance in the MDR1 gene encoding P-gp predicts absorption variability. METHODS AND RESULTS: P-gp-mediated transport of clopidogrel was assessed by transflux, influx, and efflux experiments by use of Caco-2 cells. Inhibition of P-gp activity by different modulators increased the absorptive clopidogrel flux across Caco-2 monolayers from 0.51+/-0.19 pmol/cm2 (mean+/-SD) at baseline by a maximum of 5- to 9-fold (P<.001) and the intracellular accumulation from 0.99+/-0.11 pmol/mg protein by a maximum of 2.5-fold (P<.001) in response to 1-micromol/L clopidogrel and decreased clopidogrel efflux to the level of passive diffusion. In 60 patients with coronary artery disease who underwent percutaneous coronary intervention, the peak plasma concentration (Cmax) and the total area under the plasma concentration-time curve (AUC) of clopidogrel and its active metabolite after a single oral loading dose of 300, 600, or 900 mg were tested for correlation with the MDR1 genotype. In the 300-mg and 600-mg groups (but not in the 900-mg group) Cmax and AUC values were lower in subjects homozygous for the MDR1 3435T variant compared with subjects with the 3435C/T and 3435C/C genotypes. After the 600-mg loading dose, Cmax values (mean+/-SD) of clopidogrel and its active metabolite in 3435T/T carriers were 13.3+/-5.2 ng/mL and 2.5+/-1.2 ng/mL, respectively, compared with 49.7+/-41.6 ng/mL (P=.001) and 6.6+/-3.6 ng/mL (P=.011), respectively, in 3435C/T and 3435C/C carriers; AUC values were 1502+/-463 ng/mLxmin for clopidogrel and 209+/-99 ng/mL x min for its active metabolite in 3435T/T carriers compared with 7057+/-5443 ng/mLxmin (P=.0006) and 744+/-541 ng/mLxmin (P=.011), respectively, in 3435C/T and 3435C/C carriers. CONCLUSIONS: Clopidogrel absorption and thereby active metabolite formation are diminished by P-gp-mediated efflux and are influenced by the MDR1 C3435T genotype.

Accelerated wound repair in ADAM-9 knockout animals.

ADAM-9 belongs to a family of transmembrane, disintegrin-containing metalloproteinases (ADAMs) involved in protein ectodomain shedding and cell-cell and cell-matrix interactions. Although the functions of many ADAM family members are known, the specific biological function of ADAM-9 is still unclear. In this study, we have analyzed ADAM-9 temporal and spatial distribution during wound healing. We showed increased ADAM-9 transcript expression during the first 7 days post-wounding and, by immunolocalization, detected ADAM-9 in all migrating and proliferating keratinocytes from days 3 to 7. In older 14-day-old wounds, ADAM-9 expression was restored. We have investigated the role of this protein in the healing process following excisional wounding. Animals deficient in ADAM-9 showed accelerated wound repair compared with control littermates. No alterations in neutrophil, leukocyte, and macrophage infiltration were observed. However, re-epithelialization was significantly faster in Adam-9 -/- than control wounds. Although no differences in proliferation were observed in vivo and in vitro, increased migration of keratinocytes was responsible for this effect. These results show the previously unreported role of ADAM-9 in wound repair by regulating keratinocyte migration through modulation of collagen XVII shedding.

Novel mutations of the extraneuronal monoamine transporter gene in children and adolescents with obsessive-compulsive disorder.

Obsessive-compulsive disorder (OCD) is a disease of complex aetiology with a marked genetic component. Impact of the serotonergic system has been reported but the contribution of additional transmitter systems to the pathogenesis seems likely. The extraneuronal monoamine transporter, EMT (SLC22A3), is implicated in non-neuronal termination of noradrenergic signalling in the central nervous system and a candidate gene for a variety of neuropsychiatric disorders. We conducted a case-control study of 84 Caucasian children and adolescents with OCD according to DSM-IV criteria, and healthy adults by comprehensive sequencing of the EMT gene. Additionally, targeted genotype analysis was done with patient-parent trios. Known polymorphisms and frequent haplotypes were not associated with OCD in the present sample. Transmission disequilibrium test was negative for the presumptive cryptic splice site 1233G>A polymorphism. However, we identified two novel independent mutations exclusively in affected patients. A thus far unknown -106/107delAG mutation was detected in three male patients of unaffected parents but was not prevalent in 204 healthy subjects (p=0.024). In a luciferase reporter assay the mutant allele conferred increased promoter activity by 36%. Furthermore, we describe the first non-synonymous substitution in the EMT gene, Met370Ile, in a family of affected female members that co-segregated with the disease. The residue exhibits a high degree of inter-species conservation. Heterologous expression of mutant cDNA revealed a 40% decline of transport capacity for norepinephrine. Rare mutations in the EMT gene suggest a causative or modulating role in genetic subtypes of OCD.

Identification of the endogenous key substrates of the human organic cation transporter OCT2 and their implication in function of dopaminergic neurons.

BACKGROUND: The etiology of neurodegenerative disorders, such as the accelerated loss of dopaminergic neurons in Parkinson's disease, is unclear. Current hypotheses suggest an abnormal function of the neuronal sodium-dependent dopamine transporter DAT to contribute to cell death in the dopaminergic system, but it has not been investigated whether sodium-independent amine transporters are implicated in the pathogenesis of Parkinson's disease. METHODOLOGY/PRINCIPAL FINDINGS: By the use of a novel tandem-mass spectrometry-based substrate search technique, we have shown that the dopaminergic neuromodulators histidyl-proline diketopiperazine (cyclo(his-pro)) and salsolinol were the endogenous key substrates of the sodium-independent organic cation transporter OCT2. Quantitative real-time mRNA expression analysis revealed that OCT2 in contrast to its related transporters was preferentially expressed in the dopaminergic regions of the substantia nigra where it colocalized with DAT and tyrosine hydroxylase. By assessing cell viability with the MTT reduction assay, we found that salsolinol exhibited a selective toxicity toward OCT2-expressing cells that was prevented by cyclo(his-pro). A frequent genetic variant of OCT2 with the amino acid substitution R400C reduced the transport efficiency for the cytoprotective cyclo(his-pro) and thereby increased the susceptibility to salsolinol-induced cell death. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that the OCT2-regulated interplay between cyclo(his-pro) and salsolinol is crucial for nigral cell integrity and that a shift in transport efficiency may impact the risk of Parkinson's disease.

Association of rheumatoid arthritis with ergothioneine levels in red blood cells: a case control study.

OBJECTIVE: The dietary thiol compound and erythrocyte ingredient ergothioneine (ET) is the preferential physiological substrate of the organic cation transporter OCTN1, found to be associated with rheumatoid arthritis (RA) in genetic studies, but the biological roles of ET and OCTN1 are unclear. We investigated the association between ET concentrations in peripheral blood erythrocytes and the occurrence of RA. METHODS: Erythrocyte ET concentrations in patients with mildly active RA (n = 73) were compared to ET levels in patients with coronary heart disease (CHD; n = 62) and osteoarthritis (OA; n = 148), serving as non-RA chronic inflammatory disease controls. Correlation of ET levels in erythrocytes with levels of ET and OCTN1 mRNA in CD14+ monocytes was determined in 10 healthy subjects. RESULTS: Erythrocyte ET levels were significantly higher in patients with RA, with a median (interquartile range) of 12.6 micromole/l of erythrocytes (IQR 8.1-18.3), compared to 7.7 (IQR 5.0-12.0; p < 0.001) in CHD and 7.8 (IQR 4.8-12.8; p < 0.001) in OA. The prevalence of RA compared to non-RA controls increased with increasing blood ET concentrations, with an odds ratio of 0.23 (95% CI 0.13-0.41; p < 0.001) in the lowest quartile of RA erythrocyte ET levels to 3.11 (95% CI 1.54-6.29; p = 0.002) in the highest quartile. The group differences in ET values were maintained after adjustment for disease-related anthropometric and clinical variables (age, sex, body mass index, smoking, duration of disease, hemoglobin, C-reactive protein, and medication) and were also independent of erythrocyte glutathione levels and of polymorphisms of the OCTN1 gene. ET levels in erythrocytes were linearly correlated with ET concentrations (R2 = 0.936, p < 0.001) and OCTN1 mRNA levels (R2 = 0.946, p < 0.001) in CD14+ cells. CONCLUSION: Mildly active cases of RA are associated with an unexplained high level of ET in red blood cells.