Search Results (7)

Astrocytes actively participate in neurotransmitter homeostasis by bidirectional communication with neuronal cells, a concept named the tripartite synapse, yet their role in dopamine (DA) homeostasis remains understudied. In the present study, we investigated the kinetic and molecular mechanisms of DA transport in cultured striatal astrocytes of adult rats. Kinetic uptake experiments were performed using radiolabeled [³H]-DA, whereas mRNA expression of the dopamine, norepinephrine, organic cation and plasma membrane monoamine transporters (DAT, NET, OCTs and PMAT) and DA receptors D1 and D2 was determined by qPCR. Additionally, astrocyte cultures were subjected to a 24 h treatment with the DA receptor agonist apomorphine, the DA receptor antagonist haloperidol and the DA precursor L-DOPA. [³H]-DA uptake exhibited temperature, concentration and sodium dependence, with potent inhibition by desipramine, nortriptyline and decynium-22, suggesting the involvement of multiple transporters. qPCR revealed prominent mRNA expression of the NET, the PMAT and OCT1, alongside lower levels of mRNA for OCT2, OCT3 and the DAT. Notably, apomorphine significantly altered NET, PMAT and D1 mRNA expression, while haloperidol and L-DOPA had a modest impact. Our findings demonstrate that striatal astrocytes aid in DA clearance by multiple transporters, which are influenced by dopaminergic drugs. Our study enhances the understanding of regional DA uptake, paving the way for targeted therapeutic interventions in dopaminergic disorders. Full article

Multiple drugs are used to treat various indications as well as pesticides that are ingested unintentionally and enter the bloodstream. The residence time or bioavailability of these substances in circulation depends on several mechanisms, such as drug–drug interaction (DDI), drug–pesticide interaction, metabolizing enzymes and the hepatic and renal transport systems, involved in the elimination of the compounds from the body. One of these transporters is the Organic Cation Transporter 2 (OCT2) member of the solute carrier (SLC22) transporter family. OCT2 is highly expressed in the proximal tubule epithelial cells in human and mouse kidney, where it mediates the uptake of endogenous organic cations as well as numerous drugs and xenobiotics, and contributes to the first step of renal clearance. In this study, we examined OCT2 on two subjects: First, the transferability of data from mouse to human, since mice are initially examined in the development of new drugs to assess the renal excretion of organic cations. Second, to what extent the choice of substrate affects the properties of an inhibitor. For this purpose, the functional properties of hOCT2 and mOct2 were validated under the same experimental conditions with the known substrates metformin and 1-Methyl-4-phenylpyridinium iodide (MPP). While hOCT2 and mOct2 showed very low affinities for metformin with K_m values of 3.9 mM and 3.5 mM, the affinity of hOCT2 and mOct2 for MPP (62 and 40 µM) was 64- and 89-fold higher, respectively. For our positive control inhibitor decynium22, we determined the following IC₅₀ values for hOCT2 and mOct2: 2.2 and 2.6 µM for metformin uptake, and 16 and 6.9 µM for MPP uptake. A correlation analysis of the inhibitory effects of 13 drugs and 9 pesticides on hOCT2- and mOct2-mediated transport of metformin showed a correlation coefficient R² of 0.88, indicating good interspecies correlation. Nevertheless, the bioenhancer elacridar and the fungicide imazalil showed species-dependent inhibitory potentials. Concentration-dependent inhibition of hOCT2- and mOct2-mediated metformin uptake by elacridar showed IC₅₀ values of 20 µM and 1.9 µM and by imazalil 4.7 µM and 0.58 µM, respectively. In conclusion, although our data show comparable species-independent interactions for most compounds, there can be large species–specific differences in the interactions of individual compounds, which should be considered when extrapolating data from mice to humans. Furthermore, a comparison of the inhibitory potential of elacridar and imazalil on metformin uptake with that on MPP uptake reveals substrate-dependent differences in hOCT2 and mOct2 for both inhibitors. Therefore, it might be useful to test two different substrates in inhibition studies. Full article

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. Full article

The organic cation transporters OCT1-3 (SLC22A1-3) facilitate the transport of cationic endo- and xenobiotics and are important mediators of drug distribution and elimination. Their polyspecific nature makes OCTs highly susceptible to drug–drug interactions (DDIs). Currently, screening of OCT inhibitors depends on uptake assays that require labeled substrates to detect transport activity. However, these uptake assays have several limitations. Hence, there is a need to develop novel assays to study OCT activity in a physiological relevant environment without the need to label the substrate. Here, a label-free impedance-based transport assay is established that detects OCT-mediated transport activity and inhibition utilizing the neurotoxin MPP⁺. Uptake of MPP⁺ by OCTs induced concentration-dependent changes in cellular impedance that were inhibited by decynium-22, corticosterone, and Tyrosine Kinase inhibitors. OCT-mediated MPP⁺ transport activity and inhibition were quantified on both OCT1-3 overexpressing cells and HeLa cells endogenously expressing OCT3. Moreover, the method presented here is a valuable tool to identify novel inhibitors and potential DDI partners for MPP⁺ transporting solute carrier proteins (SLCs) in general. Full article

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. Full article

Extrapolation from animal to human data is not always possible, because several essential factors, such as expression level, localization, as well as the substrate selectivity and affinity of relevant transport proteins, can differ between species. In this study, we examined the interactions of drugs and pesticides with the clinically relevant organic cation transporter hOCT1 (SLC22A1) in comparison to the orthologous transporters from mouse and rat. We determined K_m-values (73 ± 7, 36 ± 13, and 57 ± 5 µM) of human, mouse and rat OCT1 for the commonly used substrate 1-methyl-4-phenylpyridinium (MPP) and IC₅₀-values of decynium22 (12.1 ± 0.8, 5.3 ± 0.4, and 10.5 ± 0.4 µM). For the first time, we demonstrated the interaction of the cationic fungicides imazalil, azoxystrobin, prochloraz, and propamocarb with human and rodent OCT1. Drugs such as ketoconazole, clonidine, and verapamil showed substantial inhibitory potential to human, mouse, and rat OCT1 activity. A correlation analysis of hOCT1 versus mouse and rat orthologs revealed a strong functional correlation between the three species. In conclusion, this approach shows that transporter interaction data are in many cases transferable between rodents and humans, but potential species differences for other drugs and pesticides could not be excluded, though it is recommendable to perform functional comparisons of human and rodent transporters for new molecular entities. Full article

A series of four water-soluble salicylaldehyde thiosemicarbazones with a positively charged trimethylammonium moiety ([H₂L^R]Cl, R = H, Me, Et, Ph) and four copper(II) complexes [Cu(HL^R)Cl]Cl (1–4) were synthesised with the aim to study (i) their antiproliferative activity in cancer cells and, (ii) for the first time for thiosemicarbazones, the interaction with membrane transport proteins, specifically organic cation transporters OCT1–3. The compounds were comprehensively characterised by analytical, spectroscopic and X-ray diffraction methods. The highest cytotoxic effect was observed in the neuroblastoma cell line SH-5YSY after 24 h exposure and follows the rank order: 3 > 2 > 4 > cisplatin > 1 >> [H₂L^R]Cl. The copper(II) complexes showed marked interaction with OCT1–3, comparable to that of well-known OCT inhibitors (decynium 22, prazosin and corticosterone) in the cell-based radiotracer uptake assays. The work paves the way for the development of more potent and selective anticancer drugs and/or OCT inhibitors. Full article