[Show abstract][Hide abstract] ABSTRACT: Metal-binding proteins, metallothioneins (MTs), may play important roles in bone metabolism. However, the contribution of MTs to bone metabolism remains obscure. In the present study, we investigated the expression of MT isoforms in bone cells and mRNA for MT isoforms in the tibiae following ovariectomy (OVX). The results obtained showed that MT-I/II and MT-III were expressed in osteoblasts, osteoclasts, and osteocytes 4 weeks after OVX. Peaks in the mRNA expression ratios (OVX/Sham) of MT-I/II and MT-III changed following OVX. The expression ratio of MT-I/II increased after 1 week, whereas that of MT-III increased 4 weeks after OVX. These results suggest that the contribution of MTs to bone metabolism may depend on the isoforms in the cell types and the stage after OVX.
Preview · Article · Jan 2016 · Journal of Hard Tissue Biology
[Show abstract][Hide abstract] ABSTRACT: The GABAergic system in the spinal cord has been shown to participate in neuropathic pain in various animal models. GABA transporters (GATs) play a role in controlling the synaptic clearance of GABA; however, their role in neuropathic pain remains unclear. In the present study, we compared the betaine/GABA transporter (BGT-1) with other GAT subtypes to determine its participation in neuropathic pain using a mouse model of sciatic nerve ligation. 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), an inhibitor that displays moderate selectivity for BGT-1, had an antiallodynic action on model mice treated through both intrathecally and intravenous administration routes. On the other hand, SKF89976A, a selective GAT-1 inhibitor, had a weak antiallodynic action, and (S)-SNAP5114, an inhibitor that displays selectivity for GAT-3, had no antiallodynic action. Systemic analysis of these compounds on GABA uptake in CHO cells stably expressing BGT-1 revealed that NNC05-2090 not only inhibited BGT-1, but also serotonin, noradrenaline, and dopamine transporters, using a substrate uptake assay in CHO cells stably expressing each transporter, with IC50: 5.29, 7.91, and 4.08 μM, respectively. These values were similar to the IC50 value at BGT-1 (10.6 μM). These results suggest that the antiallodynic action of NNC05-2090 is due to the inhibition of both BGT-1 and monoamine transporters.
No preview · Article · May 2014 · Journal of Pharmacological Sciences
[Show abstract][Hide abstract] ABSTRACT: Aims:
Cisplatin (CDDP) is a potent anticancer agent, but severe renal toxicity can limit its use. We investigated the protective effect of cepharanthin (CE), a biscoclaurin alkaloid, on the renal toxicity of CDDP.
Mice were given CDDP along with CE. Effects of CE on CDDP toxicity were investigated by assaying markers of renal toxicity together with MT expression, and by histopathological examination of the kidney. MT-null mice were also examined.
CE induced expression of metallothionein (MT). Pre-administration of CE attenuated an increase in blood urea nitrogen (BUN) concentrations after the CDDP injection. A histochemical analysis demonstrated protection against CDDP-induced necrocytosis of kidney tissues by CE. The protective effect of CE did not occur in the MT-null mice.
Pretreatment with CE may reduce the renal toxicity of CDDP through expression of MT.
[Show abstract][Hide abstract] ABSTRACT: Betaine/γ-aminobutyric acid (GABA) transporter (BGT1, SLC6A12) is a member of the Na(+)- and Cl(-)-dependent neurotransmitter transporter gene family with a homology to the GABA transporters (GATs), GAT1 (SLC6A1), GAT2 (SLC6A13) and GAT3 (SLC6A11) (HUGO nomenclature). Since antidepressants have been reported to inhibit GABA uptake, we examined those effects on mouse BGT1 (mBGT1) in comparison with other mouse GAT (mGAT) subtypes in the heterologously expressed cell cultures. All antidepressants tested here inhibited the [(3)H]GABA uptake through mBGT1 and mGATs in a rank order of potency with mBGT1 > mGAT1-3. Kinetic analyses for maprotilline, mianserine and trimipramine revealed that they inhibited mBGT1 and mGAT1 noncompetitively, except that mianserine competitively inhibited mBGT1. These results provided a clue to investigate the structure-function relationship of mBGT1 using antidepressants as a tool, leading to the identification of potential candidates for selective and specific inhibitors of mBGT1.
Preview · Article · Dec 2012 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: The potency of anesthetics changes during development, probably due not only to pharmacokinetic factors such as differential distribution and/or metabolism, but also to pharmacodynamic factors such as changes to the GABAergic system in the brain. To explore the latter mechanism, we focused on the GABA transporter (GAT), the uptake system for GABA, which participates in the synaptic clearance of GABA. Thiopental-induced anesthesia, as assessed by the onset and duration of loss of the righting reflex, was more pronounced in 3-week-old mice than in 7-week-old mice. Both NO-711 and SKF89976A, selective GAT-1 inhibitors, significantly enhanced the anesthesia in the 7-week-old but not in the 3-week-old mice. In synaptosomes prepared from the cerebral cortex, the kinetics of GABA transport was similar between the two age groups, as assessed by [(3)H]GABA uptake assay. In addition, expression of GAT mRNA was similar between the two age groups, as assessed by quantitative RT-PCR. Thiopental reduced [(3)H]GABA uptake only at high concentrations in a similar manner at both ages. Conversely, the ability of SKF89976A to inhibit [(3)H]GABA uptake was greater in the 7-week-old mice than in the 3-week-old mice. Based on these results, GAT seems unlikely to contribute to the greater susceptibility to thiopental anesthesia in 3-week-old mice, while the increased ability of GABA uptake inhibitors to enhance thiopental-induced anesthesia in 7-week-old mice is at least partly due to higher sensitivity of GAT to the inhibitors.
No preview · Article · Sep 2012 · Pharmacology Biochemistry and Behavior
[Show abstract][Hide abstract] ABSTRACT: Methylone (2-methylamino-1-[3,4-methylenedioxyphenyl]propane-1-one) is a synthetic hallucinogenic amphetamine analog, like MDMA (3,4-methylenedioxy- methamphetamine), considered to act on monoaminergic systems. However, the psychopharmacological profile of its cytotoxicity as a consequence of monoaminergic deficits remains unclear. We examined here the effects of methylone on the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT), using a heterologous expression system in CHO cells, in association with its cytotoxicity. Methylone inhibited the activities of DAT, NET, and SERT, but not GABA transporter-1 (GAT1), in a concentration-dependent fashion with a rank order of NET > DAT > SERT. Methylone was less effective at inhibiting DAT and NET, but more effective against SERT, than was methamphetamine. Methylone alone was not toxic to cells except at high concentrations, but in combination with methamphetamine had a synergistic effect in CHO cells expressing the monoamine transporters but not in control CHO cells or cells expressing GAT1. The ability of methylone to inhibit monoamine transporter function, probably by acting as a transportable substrate, underlies the synergistic effect of methylone and methamphetamine.
Full-text · Article · Mar 2011 · DNA research: an international journal for rapid publication of reports on genes and genomes
[Show abstract][Hide abstract] ABSTRACT: The transporters for dopamine (DAT) and norepinephrine (NET) are members of the Na+- and Cl--dependent neurotransmitter transporter family SLC6. There is a line of evidence that alternative splicing results in several isoforms of neurotransmitter transporters including NET. However, its relevance to the physiology and pathology of the neurotransmitter reuptake system has not been fully elucidated.
We found novel isoforms of human DAT and NET produced by alternative splicing in human blood cells (DAT) and placenta (NET), both of which lacked the region encoded by exon 6. RT-PCR analyses showed a difference in expression between the full length (FL) and truncated isoforms in the brain and peripheral tissues, suggesting tissue-specific alternative splicing. Heterologous expression of the FL but not truncated isoforms of DAT and NET in COS-7 cells revealed transport activity. However, immunocytochemistry with confocal microscopy and a cell surface biotinylation assay demonstrated that the truncated as well as FL isoform was expressed at least in part in the plasma membrane at the cell surface, although the truncated DAT was distributed to the cell surface slower than FL DAT. A specific antibody to the C-terminus of DAT labeled the variant but not FL DAT, when cells were not treated with Triton for permeabilization, suggesting the C-terminus of the variant to be located extracellulary. Co-expression of the FL isoform with the truncated isoform in COS-7 cells resulted in a reduced uptake of substrates, indicating a dominant negative effect of the variant. Furthermore, an immunoprecipitation assay revealed physical interaction between the FL and truncated isoforms.
The unique expression and function and the proposed membrane topology of the variants suggest the importance of isoforms of catecholamine transporters in monoaminergic signaling in the brain and peripheral tissues.
[Show abstract][Hide abstract] ABSTRACT: Nicotine modulates dopaminergic activity in the central nervous system by acting on the reuptake system, including the dopamine transporter (DAT), although precisely remains unclear. Here we investigated the effect of nicotine on the transcriptional regulation of the human DAT (hDAT) gene by conducting luciferase reporter assays. Nicotine enhanced the transcription of hDAT gene constructs in transiently transfected SK-N-SH cells. Hexamethonium, a neuronal (ganglionic) nicotinic acetylcholine receptor antagonist, blocked the action of nicotine. Functional analyses placed the nicotine-responsive region -3.5 to -1.0 kb (from the transcription start site) upstream of the core promotor region. Deletion of intron 1, known as a silencer element of the hDAT gene, abolished nicotine's stimulatory effect. Nicotine failed to stimulate DAT promotor activity in non-neuronal CHO or COS-7 cells or in SK-N-AS cells, another neuronal cell line recently reported as a model for investigating DAT gene expression. These results suggest a nicotinic cholinergic mechanism to be involved in the nicotine-induced up-regulation of DAT gene expression.
No preview · Article · Feb 2010 · Neuroscience Letters
[Show abstract][Hide abstract] ABSTRACT: 5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a synthetic orally active hallucinogenic tryptamine derivative, known also as Foxy or Foxy methoxy. However, few studies have examined its effects in vitro. In the present study, we investigated the actions of 5-MeO-DIPT against monoamine neurotransmitter transporters, including the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT), using COS-7 cells heterologously expressing these transporters and rat brain synaptosomes. 5-MeO-DIPT specifically inhibited the uptake of [3H]serotonin (5-HT) by the SERT-expressing COS-7 cells and rat striatal synaptosomes in a high affinity manner at concentrations similar to those for cocaine. The effect was reversible and competitive. 5-MeO-DIPT failed to stimulate reverse transport of [3H]5-HT through SERT, while it prevented the releasing action of methamphetamine. 5-MeO-DIPT induced cell toxicity at high concentrations in COS-7 cells, and it was not influenced by the expression of SERT. These results demonstrated that 5-MeO-DIPT acts as a competitive SERT inhibitor and has an inability to cause reverse transport, underlying its serotonergic actions.
No preview · Article · May 2007 · Toxicology Letters