Involvement of GAT2/Slc6a13 in hypotaurine uptake at fetal-facing plasma membrane of syncytiotrophoblasts at mid-to-late gestation in rats and mice.

INTRODUCTION: Hypotaurine, a precursor to taurine, is known for its antioxidant properties and is prominently present in fetal plasma and the placenta. Our previous research revealed that ezrin-knockout mice experience fetal growth retardation, coinciding with reduced hypotaurine levels in fetal plasma. This study aims to elucidate the expression and role of hypotaurine transporters within the placenta. METHODS: We employed quantitative RT-PCR to measure mRNA expression of GAT transporter family members in the placenta during mid-to-late gestation. LC/MS/MS was used to analyze the distribution of hypotaurine in different placental subregions. Immunohistochemistry was utilized to examine the localization of GAT2 in mice. Placental hypotaurine uptake from fetal circulation was studied via umbilical perfusion in rats. RESULTS: Among hypotaurine transporters, GAT2 exhibited increased mRNA and protein expression in murine placenta during mid-to-late gestation. Notably, GAT2/Slc6a13 mRNA and hypotaurine were most concentrated in the labyrinth of murine placenta. In contrast, enzymes responsible for hypotaurine synthesis, such as cysteine dioxygenase, cysteine sulfinic acid decarboxylase, and 2-aminoethanethiol dioxygenase, showed minimal expression in the labyrinth. These findings suggest that GAT2 is a key determinant of hypotaurine levels in the placental labyrinth. Immunohistochemical examination unveiled that GAT2 was predominantly localized on the fetal-facing plasma membrane within syncytiotrophoblasts, which co-localized with ezrin. In rat umbilical perfusion experiments, the GAT2/3 and TauT inhibitor, SNAP-5114, significantly reduced hypotaurine extraction from fetal circulation to the placenta. DISCUSSION: The results suggest that GAT2 plays a pivotal role in the concentrative uptake of hypotaurine from fetal plasma within syncytiotrophoblasts of the placenta.

The Use of Carboxyfluorescein Reveals the Transport Function of MCT6/SLC16A5 Associated with CD147 as a Chloride-Sensitive Organic Anion Transporter in Mammalian Cells.

Oral drug absorption involves drug permeation across the apical and basolateral membranes of enterocytes. Although transporters mediating the influx of anionic drugs in the apical membranes have been identified, transporters responsible for efflux in the basolateral membranes remain unclear. Monocarboxylate transporter 6 (MCT6/SLC16A5) has been reported to localize to the apical and basolateral membranes of human enterocytes and to transport organic anions such as bumetanide and nateglinide in the Xenopus oocyte expression system; however, its transport functions have not been elucidated in detail. In this study, we characterized the function of MCT6 expressed in HEK293T cells and explored fluorescent probes to more easily evaluate MCT6 function. The results illustrated that MCT6 interacts with CD147 to localize at the plasma membrane. When the uptake of various fluorescein derivatives was examined in NaCl-free uptake buffer (pH 5.5), the uptake of 5-carboxyfluorescein (5-CF) was significantly greater in MCT6 and CD147-expressing cells. MCT6-mediated 5-CF uptake was saturable with a Km of 1.07 mM and inhibited by several substrates/inhibitors of organic anion transporters and extracellular Cl ion with an IC50 of 53.7 mM. These results suggest that MCT6 is a chloride-sensitive organic anion transporter that can be characterized using 5-CF as a fluorescent probe.

Macrolide and Ketolide Antibiotics Inhibit the Cytotoxic Effect of Trastuzumab Emtansine in HER2-Positive Breast Cancer Cells: Implication of a Potential Drug-ADC Interaction in Cancer Chemotherapy.

Macrolides are widely used for the long-term treatment of infections and chronic inflammatory diseases. The pharmacokinetic features of macrolides include extensive tissue distribution because of favorable membrane permeability and accumulation within lysosomes. Trastuzumab emtansine (T-DM1), a HER2-targeting antibody-drug conjugate (ADC), is catabolized in the lysosomes, where Lys-SMCC-DM1, a potent cytotoxic agent, is processed by proteinase degradation and subsequently released from the lysosomes to the cytoplasm through the lysosomal membrane transporter SLC46A3, resulting in an antitumor effect. We recently demonstrated that erythromycin and clarithromycin inhibit SLC46A3 and attenuate the cytotoxicity of T-DM1; however, the effect of other macrolides and ketolides has not been determined. In this study, we evaluated the effect of macrolide and ketolide antibiotics on T-DM1 cytotoxicity in a human breast cancer cell line, KPL-4. Macrolides used in the clinic, such as roxithromycin, azithromycin, and josamycin, as well as solithromycin, a ketolide under clinical development, significantly attenuated T-DM1 cytotoxicity in addition to erythromycin and clarithromycin. Of these, azithromycin was the most potent inhibitor of T-DM1 efficacy. These antibiotics significantly inhibited the transport function of SLC46A3 in a concentration-dependent manner. Moreover, these compounds extensively accumulated in the lysosomes at the levels estimated to be 0.41-13.6 mM when cells were incubated with them at a 2 µM concentration. The immunofluorescence staining of trastuzumab revealed that azithromycin and solithromycin inhibit the degradation of T-DM1 in the lysosomes. These results suggest that the attenuation of T-DM1 cytotoxicity by macrolide and ketolide antibiotics involves their lysosomal accumulation and results in their greater lysosomal concentrations to inhibit the SLC46A3 function and T-DM1 degradation. This suggests a potential drug-ADC interaction during cancer chemotherapy.

Monocarboxylate Transporter 13 (MCT13/SLC16A13) Functions as a Novel Plasma Membrane Oligopeptide Transporter.

SLC16A13, which encodes the monocarboxylate transporter 13 (MCT13), is a susceptibility gene for type 2 diabetes and is expressed in the liver and duodenum. Some peptidase-resistant oligopeptides are absorbed in the gastrointestinal tract and affect glycemic control in the body. Their efficient absorption is mediated by oligopeptide transporter(s) at the apical and basolateral membranes of the intestinal epithelia; however, the molecules responsible for basolateral oligopeptide transport have not been identified. In this study, we examined whether MCT13 functions as a novel basolateral oligopeptide transporter. We evaluated the uptake of oligopeptides and peptidomimetics in MCT13-transfected cells. The uptake of cephradine, a probe for peptide transport system(s), significantly increased in MCT13-transfected cells, and this increase was sensitive to membrane potential. The cellular accumulation of bioactive peptides, such as anserine and carnosine, was decreased by MCT13, indicating MCT13-mediated efflux transport activity. In polarized Caco-2 cells, MCT13 was localized at the basolateral membrane. MCT13 induction enhanced cephradine transport in an apical-to-basal direction across Caco-2 cells. These results indicate that MCT13 functions as a novel efflux transporter of oligopeptides and peptidomimetics, driven by electrochemical gradients across the plasma membrane, and it may be involved in the transport of these compounds across the intestinal epithelia.

The Glycosylated N-Terminal Domain of MUC1 Is Involved in Chemoresistance by Modulating Drug Permeation Across the Plasma Membrane.

Mucin 1 (MUC1) is aberrantly expressed in various cancers and implicated in cancer progression and chemoresistance. Although the C-terminal cytoplasmic tail of MUC1 is involved in signal transduction, promoting chemoresistance, the role of the extracellular MUC1 domain [N-terminal glycosylated domain (NG)-MUC1] remains unclear. In this study, we generated stable MCF7 cell lines expressing MUC1 and cytoplasmic tail-deficient MUC1 (MUC1ΔCT) and show that NG-MUC1 is involved in drug resistance by modulating the transmembrane permeation of various compounds without cytoplasmic tail signaling. Heterologous expression of MUC1ΔCT increased cell survival in treating anticancer drugs (such as 5-fluorouracil, cisplatin, doxorubicin, and paclitaxel), in particular by causing an approximately 150-fold increase in the IC50 of paclitaxel, a lipophilic drug, compared with the control [5-fluorouracil (7-fold), cisplatin (3-fold), and doxorubicin (18-fold)]. The uptake studies revealed that accumulations of paclitaxel and Hoechst 33342, a membrane-permeable nuclear staining dye, were reduced to 51% and 45%, respectively, in cells expressing MUC1ΔCT via ABCB1/P-gp-independent mechanisms. Such alterations in chemoresistance and cellular accumulation were not observed in MUC13-expressing cells. Furthermore, we found that MUC1 and MUC1ΔCT increased the cell-adhered water volume by 2.6- and 2.7-fold, respectively, suggesting the presence of a water layer on the cell surface created by NG-MUC1. Taken together, these results suggest that NG-MUC1 acts as a hydrophilic barrier element against anticancer drugs and contributes to chemoresistance by limiting the membrane permeation of lipophilic drugs. Our findings could help better the understanding of the molecular basis of drug resistance in cancer chemotherapy. SIGNIFICANCE STATEMENT: Membrane-bound mucin (MUC1), aberrantly expressed in various cancers, is implicated in cancer progression and chemoresistance. Although the MUC1 cytoplasmic tail is involved in proliferation-promoting signal transduction thereby leading to chemoresistance, the significance of the extracellular domain remains unclear. This study clarifies the role of the glycosylated extracellular domain as a hydrophilic barrier element to limit the cellular uptake of lipophilic anticancer drugs. These findings could help better the understanding of the molecular basis of MUC1 and drug resistance in cancer chemotherapy.

Identification of 5-Carboxyfluorescein as a Probe Substrate of SLC46A3 and Its Application in a Fluorescence-Based In Vitro Assay Evaluating the Interaction with SLC46A3.

The therapeutic modalities that involve the endocytosis pathway, including antibody-drug conjugates (ADCs), have recently been developed. Since the drug escape from endosomes/lysosomes is a determinant of their efficacy, it is important to optimize the escape, and the cellular evaluation system is needed. SLC46A3, a lysosomal membrane protein, has been implicated in the pharmacological efficacy of trastuzumab emtansine (T-DM1), a noncleavable ADC used for the treatment of breast cancer, and the cellular uptake efficacy of lipid-based nanoparticles. Recently, we identified the SLC46A3 function as a proton-coupled steroid conjugate and bile acid transporter, which can directly transport active catabolites of T-DM1. Thus, the rapid and convenient assay systems for evaluating the SLC46A3 function may help to facilitate ADC development and to clarify the physiological roles in endocytosis. Here, we show that SLC46A3 dC, which localizes to the plasma membrane owing to lacking a lysosomal-sorting motif, has a great ability to transport 5-carboxyfluorescein (5-CF), a fluorescent probe, in a pH-dependent manner. 5-CF uptake mediated by SLC46A3 was significantly inhibited by compounds reported to be SLC46A3 substrates/inhibitors and competitively inhibited by estrone 3-sulfate, a typical SLC46A3 substrate. The inhibition assays followed by uptake studies revealed that SG3199, a pyrrolobenzodiazepine dimer, which has been used as an ADC payload, is a substrate of SLC46A3. Accordingly, the fluorescence-based assay system for the SLC46A3 function using 5-CF can provide a valuable tool to evaluate the interaction of drugs/drug candidates with SLC46A3.

Naltrexone Transport by a Proton-Coupled Organic Cation Antiporter in hCMEC/D3 Cells, an in Vitro Human Blood-Brain Barrier Model.

Naltrexone is a mu-opioid receptor antagonist used in the treatment of opioid and alcohol dependence. The blood-brain barrier (BBB) transport characteristics of naltrexone was investigated by means of hCMEC/D3 cells, a human immortalized brain capillary endothelial cell line. In hCMEC/D3 cells, naltrexone is taken up in a concentration-dependent manner. Furthermore, naltrexone uptake significantly decreased in the presence of H+/organic cation (OC) antiporter substrates, during the little alteration exhibited by substrates of well-identified OC transporters classified into SLC22A family. Although naltrexone uptake by hCMEC/D3 cells was partially affected by changes of ionic conditions, it was markedly decreased in the presence of the metabolic inhibitor sodium azide. Furthermore, when treated by ammonium chloride, naltrexone uptake by hCMEC/D3 cells was altered by intracellular acidification and alkalization, suggesting the involvement of oppositely directed proton gradient in naltrexone transport across the BBB. The results obtained in the present in vitro study suggest the active transport of naltrexone from blood to the brain across the BBB by the H+/OC antiporter.

Proteomics-Based Transporter Identification by the PICK Method: Involvement of TM7SF3 and LHFPL6 in Proton-Coupled Organic Cation Antiport at the Blood-Brain Barrier.

A proton-coupled organic cation (H+/OC) antiporter working at the blood-brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the Proteomics-based Identification of transporter by Crosslinking substrate in Keyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H+/OC antiporter. We synthesized a novel H+/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H+/OC antiporter.

Bright and Light-Up Sensing of Benzo[c,d]indole-oxazolopyridine Cyanine Dye for RNA and Its Application to Highly Sensitive Imaging of Nucleolar RNA in Living Cells.

Small molecular weight probes that can show a fluorescence signaling response upon binding to RNAs are promising for RNA imaging in living cells. Live-cell RNA imaging probes that can achieve a large light-up ability (>100-fold) and high Φbound value for RNA (>0.50) have been rarely reported to date. Here, benzo[c,d]indole-oxazolopyridine (BIOP), an unsymmetrical monomethine cyanine analogue, was newly developed as a bright and large light-up probe for imaging of nucleolar RNA in living cells. BIOP served as a yellow-emissive probe (λem = 570 nm) and exhibited a significant light-up response upon RNA binding (770-fold) with a high Φbound value (0.52). We demonstrated the advantages of BIOP over a commercially available RNA-staining probe, SYTO RNA select, for robust and sensitive RNA sensing by a systematic comparison of fluorescent properties for RNA. In addition, BIOP was found to possess high membrane permeability and low cytotoxicity in living cells. The examination of live-cell imaging revealed that BIOP exhibited emission in the nucleolus upon binding to nucleolar RNA much stronger than that of SYTO RNA select. Furthermore, BIOP facilitated the highly sensitive imaging of nucleolar RNA, in which 50 nM BIOP can stain nucleolar RNA in living cells with a 20 min incubation.

Binding of Citrate-Fe3+ to Plastic Culture Dishes, an Artefact Useful as a Simple Technique to Screen for New Iron Chelators.

NaCT mediates citrate uptake in the liver cell line HepG2. When these cells were exposed to iron (Fe3+), citrate uptake/binding as monitored by the association of [14C]-citrate with cells increased. However, there was no change in NaCT expression and function, indicating that NaCT was not responsible for this Fe3+-induced citrate uptake/binding. Interestingly however, the process exhibited substrate selectivity and saturability as if the process was mediated by a transporter. Notwithstanding these features, subsequent studies demonstrated that the iron-induced citrate uptake/binding did not involve citrate entry into cells; instead, the increase was due to the formation of citrate-Fe3+ chelate that adsorbed to the cell surface. Surprisingly, the same phenomenon was observed in culture wells without HepG2 cells, indicating the adsorption of the citrate-Fe3+ chelate to the plastic surface of culture wells. We used this interesting phenomenon as a simple screening technique for new iron chelators with the logic that if another iron chelator is present in the assay system, it would compete with citrate for binding to Fe3+ and prevent the formation and adsorption of citrate-Fe3+ to the culture well. This technique was validated with the known iron chelators deferiprone and deferoxamine, and with the bacterial siderophore 2,3-dihydroxybenzoic acid and the catechol carbidopa.

Mammalian monocarboxylate transporter 7 (MCT7/Slc16a6) is a novel facilitative taurine transporter.

Monocarboxylate transporter 7 (MCT7) is an orphan transporter expressed in the liver, brain, and in several types of cancer cells. It has also been reported to be a survival factor in melanoma and breast cancers. However, this survival mechanism is not yet fully understood due to MCT7's unidentified substrate(s). Therefore, here we sought to identify MCT7 substrate(s) and characterize the transport mechanisms by analyzing amino acid transport in HEK293T cells and polarized Caco-2 cells. Analysis of amino acids revealed significant rapid reduction in taurine from cells transfected with enhanced green fluorescent protein-tagged MCT7. We found that taurine uptake and efflux by MCT7 was pH-independent and that the uptake was not saturated in the presence of taurine excess of 200 mM. Furthermore, we found that monocarboxylates and acidic amino acids inhibited MCT7-mediated taurine uptake. These results imply that MCT7 may be a low-affinity facilitative taurine transporter. We also found that MCT7 was localized at the basolateral membrane in polarized Caco-2 cells and that the induction of MCT7 expression in polarized Caco-2 cells enhanced taurine permeation. Finally, we demonstrated that interactions of MCT7 with ancillary proteins basigin/CD147 and embigin/GP70 enhanced MCT7-mediated taurine transport. In summary, these findings reveal that taurine is a novel substrate of MCT7 and that MCT7-mediated taurine transport might contribute to the efflux of taurine from cells.

SLC46A3 is a lysosomal proton-coupled steroid conjugate and bile acid transporter involved in transport of active catabolites of T-DM1.

Antibody-drug conjugates (ADCs) represent a new class of cancer therapeutics that enable targeted delivery of cytotoxic drugs to cancer cells. Although clinical efficacy has been demonstrated for ADC therapies, resistance to these conjugates may occur. Recently, SLC46A3, a lysosomal membrane protein, was revealed to regulate the efficacy of trastuzumab emtansine (T-DM1), a noncleavable ADC that has been widely used for treating breast cancer. However, the role of SLC46A3 in mediating T-DM1 cytotoxicity remains unclear. In this study, we discovered the function of SLC46A3 as a novel proton-coupled steroid conjugate and bile acid transporter. SLC46A3 preferentially recognized lipophilic steroid conjugates and bile acids as endogenous substrates. In addition, we found that SLC46A3 directly transports Lys-SMCC-DM1, a major catabolite of T-DM1, and potent SLC46A3 inhibitors attenuate the cytotoxic effects of T-DM1, suggesting a role in the escape of Lys-SMCC-DM1 from the lysosome into the cytoplasm. Our findings reveal the molecular mechanism by which T-DM1 kills cancer cells and may contribute to the rational development of ADCs that target SLC46A3.

A Proton-Coupled Transport System for ß-Hydroxy-ß-Methylbutyrate (HMB) in Blood-Brain Barrier Endothelial Cell Line hCMEC/D3.

ß-Hydroxy-ß-methylbutyrate (HMB), a leucine metabolite, is used as a nutritional ingredient to improve skeletal muscle health. Preclinical studies indicate that this supplement also elicits significant benefits in the brain; it promotes neurite outgrowth and prevents age-related reductions in neuronal dendrites and cognitive performance. As orally administered HMB elicits these effects in the brain, we infer that HMB crosses the blood-brain barrier (BBB). However, there have been no reports detailing the transport mechanism for HMB in BBB. Here we show that HMB is taken up in the human BBB endothelial cell line hCMEC/D3 via H+-coupled monocarboxylate transporters that also transport lactate and ß-hydroxybutyrate. MCT1 (monocarboxylate transporter 1) and MCT4 (monocarboxylate transporter 4) belonging to the solute carrier gene family SLC16 (solute carrier, gene family 16) are involved, but additional transporters also contribute to the process. HMB uptake in BBB endothelial cells results in intracellular acidification, demonstrating cotransport with H+. Since HMB is known to activate mTOR with potential to elicit transcriptomic changes, we examined the influence of HMB on the expression of selective transporters. We found no change in MCT1 and MCT4 expression. Interestingly, the expression of LAT1 (system L amino acid transporter 1), a high-affinity transporter for branched-chain amino acids relevant to neurological disorders such as autism, is induced. This effect is dependent on mTOR (mechanistic target of rapamycine) activation by HMB with no involvement of histone deacetylases. These studies show that HMB in systemic circulation can cross the BBB via carrier-mediated processes, and that it also has a positive influence on the expression of LAT1, an important amino acid transporter in the BBB.

Deep-red fluorogenic cyanine dyes carrying an amino group-terminated side chain for improved RNA detection and nucleolar RNA imaging.

The introduction of an amino-group-terminated side chain into deep-red emissive benzo[c,d]indole-quinoline monomethine cyanine dye has led to the improved detection of RNAs as well as the imaging of nucleolar RNAs in cells.

Functional Investigation of Solute Carrier Family 35, Member F2, in Three Cellular Models of the Primate Blood-Brain Barrier.

Understanding the mechanisms of drug transport across the blood-brain barrier (BBB) is an important issue for regulating the pharmacokinetics of drugs in the central nervous system. In this study, we focused on solute carrier family 35, member F2 (SLC35F2), whose mRNA is highly expressed in the BBB. SLC35F2 protein was enriched in isolated mouse and monkey brain capillaries relative to brain homogenates and was localized exclusively on the apical membrane of MDCKII cells and brain microvascular endothelial cells (BMECs) differentiated from human induced pluripotent stem cells (hiPS-BMECs). SLC35F2 activity was assessed using its substrate, YM155, and pharmacological experiments revealed SLC35F2 inhibitors, such as famotidine (half-maximal inhibitory concentration, 160 µM). Uptake of YM155 was decreased by famotidine or SLC35F2 knockdown in immortalized human BMECs (human cerebral microvascular endothelial cell/D3 cells). Furthermore, famotidine significantly inhibited the apical (A)-to-basal (B) transport of YM155 in primary cultured monkey BMECs and hiPS-BMECs. Crucially, SLC35F2 knockout diminished the A-to-B transport and intracellular accumulation of YM155 in hiPS-BMECs. By contrast, in studies using an in situ brain perfusion technique, neither deletion of Slc35f2 nor famotidine reduced brain uptake of YM155, even though YM155 is a substrate of mouse SLC35F2. YM155 uptake was decreased significantly by losartan and naringin, inhibitors for the organic anion transporting polypeptide (OATP) 1A4. These findings suggest SLC35F2 is a functional transporter in various cellular models of the primate BBB that delivers its substrates to the brain and that its relative importance in the BBB is modified by differences in the expression of OATPs between primates and rodents. SIGNIFICANCE STATEMENT: This study demonstrated that SLC35F2 is a functional drug influx transporter in three different cellular models of the primate blood-brain barrier (i.e., human cerebral microvascular endothelial cell/D3 cells, primary cultured monkey BMECs, and human induced pluripotent stem-BMECs) but has limited roles in mouse brain. SLC35F2 facilitates apical-to-basal transport across the tight cell monolayer. These findings will contribute to the development of improved strategies for targeting drugs to the central nervous system.

Functional analysis of a species-specific inhibitor selective for human Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY).

The Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY) in the liver delivers citrate from the blood into hepatocytes. As citrate is a key metabolite and regulator of multiple biochemical pathways, deletion of Slc13a5 in mice protects against diet-induced obesity, diabetes, and metabolic syndrome. Silencing the transporter suppresses hepatocellular carcinoma. Therefore, selective blockers of NaCT hold the potential to treat various diseases. Here we report on the characteristics of one such inhibitor, BI01383298. It is known that BI01383298 is a high-affinity inhibitor selective for human NaCT with no effect on mouse NaCT. Here we show that this compound is an irreversible and non-competitive inhibitor of human NaCT, thus describing the first irreversible inhibitor for this transporter. The mouse NaCT is not affected by this compound. The inhibition of human NaCT by BI01383298 is evident for the constitutively expressed transporter in HepG2 cells and for the ectopically expressed human NaCT in HEK293 cells. The IC50 is ∼100 nM, representing the highest potency among the NaCT inhibitors known to date. Exposure of HepG2 cells to this inhibitor results in decreased cell proliferation. We performed molecular modeling of the 3D-structures of human and mouse NaCTs using the crystal structure of a humanized variant of VcINDY as the template, and docking studies to identify the amino acid residues involved in the binding of citrate and BI01383298. These studies provide insight into the probable bases for the differential effects of the inhibitor on human NaCT versus mouse NaCT as well as for the marked species-specific difference in citrate affinity.

Involvement of a Na+-coupled Oligopeptide Transport System for ß-amyloid Peptide (Aß1-42) in Brain Cells.

PURPOSE: A Na+-coupled transport system in mammalian cells is responsible for the uptake of oligopeptides consisting of 5 or more amino acids. Here we investigated if this transport system is expressed in brain cells and transports the 42-amino-acid ß-amyloid peptide (Aß1-42). METHODS: The human and mouse neuronal cell lines SK-N-SH and HT22, human microglial cell line HMC-3, and human blood-brain barrier endothelial cell line hCMEC/D3 were used to monitor the uptake of [3H]-deltorphin II (a heptapeptide) and fluorescence-labeled Aß1-42. RESULTS: All four cell lines exhibited Na+-coupled uptake of deltorphin II. Aß1-42 competed with deltorphin II for the uptake. Uptake of fluorescence-labeled Aß1-42 was detectable in these cell lines, and the uptake was Na+-dependent and inhibitable by deltorphin II. The Na+-coupled uptake disappeared at high concentrations of Aß1-42 due to oligomerization of the peptide. Exposure of the cells to excess iron abolished the uptake. In hCMEC/D3 cells cultured on Transwell filters, the uptake was localized preferentially to the abluminal membrane. CONCLUSION: A Na+-coupled transport system mediates the uptake of Aß1-42 monomers in neuronal and microglial cells. The same system is also responsible for the uptake of Aß1-42 from brain into blood-brain barrier endothelial cells. These findings have relevance to Alzheimer's disease.

The Hepatic Plasma Membrane Citrate Transporter NaCT (SLC13A5) as a Molecular Target for Metformin.

Metformin is the first-line treatment for type 2 diabetes. Inhibition of hepatic gluconeogenesis is the primary contributor to its anti-diabetic effect. Metformin inhibits complex I and α-glycerophosphate shuttle, and the resultant increase in cytoplasmic NADH/NAD+ ratio diverts glucose precursors away from gluconeogenesis. These actions depend on metformin-mediated activation of AMP kinase (AMPK). Here we report on a hitherto unknown mechanism. Metformin inhibits the expression of the plasma membrane citrate transporter NaCT in HepG2 cells and decreases cellular levels of citrate. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, elicits a similar effect. The process involves a decrease in maximal velocity with no change in substrate affinity. The decrease in NaCT expression is associated with decreased mRNA levels. AMPK inhibits mTOR, and the mTOR inhibitor rapamycin also decreases NaCT expression. The transcription factor downstream of AMPK that is relevant to cAMP signaling is CREB; decreased levels of phospho-CREB seem to mediate the observed effects of metformin on NaCT. Citrate is known to suppress glycolysis by inhibiting phosphofructokinase-1 and activate gluconeogenesis by stimulating fructose-1,6-bisphophatase; therefore, the decrease in cellular levels of citrate would stimulate glycolysis and inhibit gluconeogenesis. These studies uncover a novel mechanism for the anti-diabetic actions of metformin.

Strong Binding and Off-On Signaling Functions of Deep-Red Fluorescent TO-PRO-3 for Influenza†A Virus RNA Promoter Region.

The RNA promoter region of the influenza A virus has recently attracted much attention as an RNA target for the development of anti-influenza drugs. However, there are very few reports on small RNA-binding ligands targeting this region. In this work, it is reported that TO-PRO-3, a thiazole orange analogue with a trimethine bridge, exhibits strong and selective binding to the internal loop structure of the influenza A virus RNA promoter. This binding accompanies the remarkable light-up response of TO-PRO-3 in the deep-red spectral region. By virtue of these binding and fluorescence signaling functions, TO-PRO-3 can act as a useful indicator for the assessment of the binding capabilities of various test compounds for this RNA region, with a view toward the development of anti-influenza drug candidates.

Development and radiosynthesis of the first 18 F-labeled inhibitor of monocarboxylate transporters (MCTs).

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumor development and progression. Their expression levels are related to clinical disease prognosis. Accordingly, both MCTs are promising drug targets for treatment of a variety of human cancers. The noninvasive imaging of these MCTs in cancers is regarded to be advantageous for assessing MCT-mediated effects on chemotherapy and radiosensitization using specific MCT inhibitors. Herein, we describe a method for the radiosynthesis of [18 F]FACH ((E)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylic acid), as a novel radiolabeled MCT1/4 inhibitor for imaging with PET. A fluorinated analog of α-cyano-4-hydroxycinnamic acid (FACH) was synthesized, and the inhibition of MCT1 and MCT4 was measured via an L-[14 C]lactate uptake assay. Radiolabeling was performed by a two-step protocol comprising the radiosynthesis of the intermediate (E)/(Z)-[18 F]tert-Bu-FACH (tert-butyl (E)/(Z)-2-cyano-3-{4-[(3-[18 F]fluoropropyl)(propyl)amino]-2-methoxyphenyl}acrylate) followed by deprotection of the tert-butyl group. The radiofluorination was successfully implemented using either K[18 F]F-K2.2.2 -carbonate or [18 F]TBAF. The final deprotected product [18 F]FACH was only obtained when [18 F]tert-Bu-FACH was formed by the latter procedure. After optimization of the deprotection reaction, [18 F]FACH was obtained in high radiochemical yields (39.6 ± 8.3%, end of bombardment (EOB) and radiochemical purity (greater than 98%).