Monocarboxylate Transporters 1 and 2 Are Responsible for L-Lactate Uptake in Differentiated Human Neuroblastoma SH-SY5Y Cells.

L-Lactate transport via monocarboxylate transporters (MCTs) in the central nervous system, represented by the astrocyte-neuron lactate shuttle (ANLS), is crucial for the maintenance of brain functions, including memory formation. Previously, we have reported that MCT1 contributes to L-lactate transport in normal human astrocytes. Therefore, in this study, we aimed to identify transporters that contribute to L-lactate transport in human neurons. SH-SY5Y cells, which are used as a model for human neurons, were differentiated using all-trans-retinoic acid. L-Lactate uptake was measured using radiolabeled L-lactate, and the expression of MCT proteins was confirmed Western blotting. L-Lactate transport was pH-dependent and saturated at high concentrations. Kinetic analysis suggested that L-lactate uptake was biphasic. Furthermore, MCT1, 2 selective inhibitors inhibited L-lactate transport. In addition, the expression of MCT1 and 2 proteins, but not MCT4, was confirmed. In this study, we demonstrated that MCT1 and 2 are major contributors to L-lactate transport in differentiated human neuroblastoma SH-SY5Y cells from the viewpoint of kinetic analysis. These results lead to a better understanding of ANLS in humans, and further exploration of the factors that can promote MCT1 and 2 functions is required.

Breast milk concentrations of acetaminophen and diclofenac - unexpectedly high mammary transfer of the general-purpose drug acetaminophen.

BACKGROUND: Breastfeeding is considered to be the most effective way of ensuring the health and survival of newborns. However, mammary transfer of drugs administered to mothers to breastfeeding infants remains a pressing concern. Acetaminophen and diclofenac sodium are widely prescribed analgesics for postpartum pain relief, but there have been few recent reports on the mammary transfer of these drugs, despite advances in analytic techniques. METHODS: We conducted a study on 20 postpartum mothers from August 2019-March 2020. Blood and milk samples from participants were analyzed using liquid chromatography-electrospray ionization tandem mass spectrometry within 24 hours after oral administration of acetaminophen and diclofenac sodium. The area under the concentration-time curve (AUC) was calculated from the concentration curve obtained by a naive pooled-data approach. RESULTS: For acetaminophen, AUC was 36,053 ng/mL.h and 37,768 ng/mL.h in plasma and breast milk, respectively, with a milk-to-plasma drug concentration ratio of 1.048. For diclofenac, the AUC was 0.227 ng/mL.h and 0.021 ng/mL.h, in plasma and breast milk, respectively, with a milk-to-plasma drug concentration ratio of 0.093. CONCLUSIONS: While diclofenac sodium showed low mammary transfer, acetaminophen showed a relatively high milk-to-plasma drug concentration ratio. Given recent studies suggesting potential connections between acetaminophen use during pregnancy and risks to developmental prognosis in children, we believe that adequate information regarding the fact that acetaminophen is easily transferred to breast milk should be provided to mothers.

Effects of valproic acid on syncytialization in human placental trophoblast cell lines.

The placenta is a critical organ for fetal development and a healthy pregnancy, and has multifaceted functions (e.g., substance exchange and hormone secretion). Syncytialization of trophoblasts is important for maintaining placental functions. Epilepsy is one of the most common neurological conditions worldwide. Therefore, this study aimed to reveal the influence of antiepileptic drugs, including valproic acid (VPA), carbamazepine, lamotrigine, gabapentin, levetiracetam, topiramate, lacosamide, and clobazam, at clinically relevant concentrations on syncytialization using in vitro models of trophoblasts. To induce differentiation into syncytiotrophoblast-like cells, BeWo cells were treated with forskolin. Exposure to VPA was found to dose-dependently influence syncytialization-associated genes (ERVW-1, ERVFRD-1, GJA1, CGB, CSH, SLC1A5, and ABCC4) in differentiated BeWo cells. Herein, the biomarkers between differentiated BeWo cells and the human trophoblast stem model (TSCT) were compared. In particular, MFSD2A levels were low in BeWo cells but abundant in TSCT cells. VPA exposure affected the expression of ERVW-1, ERVFRD-1, GJA1, CSH, MFSD2A, and ABCC4 in differentiated cells (ST-TSCT). Furthermore, VPA exposure attenuated BeWo and TSCT cell fusion. Finally, the relationships between neonatal/placental parameters and the expression of syncytialization markers in human term placentas were analyzed. MFSD2A expression was positively correlated with neonatal body weight, head circumference, chest circumference, and placental weight. Our findings have important implications for better understanding the mechanisms of toxicity of antiepileptic drugs and predicting the risks to placental and fetal development.

Development of a risk prediction model for surgical site infection after lower third molar surgery.

BACKGROUND: There is little evidence regarding risk prediction for surgical site infection (SSI) after lower third molar (L3M) surgery. METHODS: We conducted a nested case-control study to develop a multivariable logistic model for predicting the risk of SSI after L3M surgery. Data were obtained from Hokkaido University Hospital from April 2013 to March 2020. Multiple imputation was applied for the missing values. We conducted decision tree (DT) analysis to evaluate the combinations of factors affecting SSI risk. RESULTS: We identified 648 patients. The final model retained the available distal space (Pell & Gregory II [p = 0.05], Pell & Gregory III [p < 0.01]), depth (Pell & Gregory B [p < 0.01], Pell & Gregory C [p < 0.01]), surgeon's experience (3-10 years [p = 0.25], <3 years [p < 0.01]), and simultaneous extraction of both L3M [p < 0.01]; the concordance-statistic was 0.72. The DT analysis demonstrated that patients with Pell and Gregory B or C and simultaneous extraction of both L3M had the highest risk of SSI. CONCLUSIONS: We developed a model for predicting SSI after L3M surgery with adequate predictive metrics in a single center. This model will make the SSI risk prediction more accessible.

Association between α-defensin 5 and the expression and function of P-glycoprotein in differentiated intestinal Caco-2 cells.

α-Defensin 5 is known to be secreted by Paneth cells in the small intestine and plays an important role in eliminating pathogenic microorganisms. It has been reported that a decrease in α-defensin 5 level in the human small intestine is a risk of inflammatory bowel disease (IBD). Furthermore, P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter superfamily, encoded by the ABCB1/MDR1 gene, plays an important role in the front line of host defense by protecting the gastrointestinal barrier from xenobiotic accumulation and may contribute to the development and persistence of IBD. Therefore, we examined the relationship between α-defensin 5 and the expression and function of P-gp using a human gastrointestinal model cell line (Caco-2). We found that MDR1 mRNA and P-gp protein level were increased in Caco-2 cells as well as α-defensin 5 secretion corresponded with the duration of cell culture. Exposure to α-defensin 5 peptide and recombinant tumor necrosis factor-α (TNF-α) significantly increased the expression and function P-gp. The mRNA levels of interleukin (IL)-8, IL-6, TNF-α, IL-1ß, and IL-2 were also increased following exposure to TNF-α, similar to α-defensin 5 treatment. These results suggest that α-defensin 5 regulates P-gp expression and function by increasing TNF-α expression in Caco-2 cells.

The rs35217482 (T755I) single-nucleotide polymorphism in aldehyde oxidase-1 attenuates prot ein dimer formation and reduces the rates of phthalazine metabolism.

Aldehyde oxidase 1 (AOX1) is a molybdenum-containing enzyme that catalyzes the oxidation of a range of aldehyde compounds and clinical drugs, including azathioprine and methotrexate. The purpose of this study was to elucidate the effects of single-nucleotide polymorphisms (SNPs) in the coding regions of the human AOX1 gene on protein dimer formation and metabolic activity. Six variants (Q314R [rs58185012], I598N [rs143935618], T755I [rs35217482], A1083G [rs139092129], N1135S [rs55754655], and H1297R [rs3731722]), with allele frequencies greater than 0.01 in 1 or more population, were obtained from the genome aggregation and 1000 Genomes project databases. Protein expression and dimer formation were evaluated using HEK293T cells expressing the wild-type (WT) or different SNP variants of AOX1. Kinetic analyses of phthalazine oxidation were performed using S9 fractions of HEK293T cells expressing WT or each the different mutant AOX1. Although we detected no significant differences among WT AOX1 and the different variants with respect to total protein expression, native PAGE analysis indicated that one of the SNP variants, T755I, found in East Asian populations, dimerizes less efficiently than the WT AOX1. Kinetic analysis, using phthalazine as a typical substrate, revealed that this mutation contributes to a reduction in the maximal rates of reaction without affecting enzyme affinity for phthalazine. Our observation thus indicates that the T755I variant has significantly negative effects on both the dimer formation and in vitro catalytic activity of AOX1. These findings may provide valuable insights into the mechanisms underlying the inter-individual differences in the therapeutic efficacy or toxicity of AOX1 substrate drugs. Significance Statement The T755l (rs35217482) SNP variant of the AOX1 protein, which is prominent in East Asian populations, suppresses protein dimer formation, resulting in a reduction in the reaction velocity of phthalazine oxidation to less than half of that of wild-type AOX1.

Uptake of antiepileptic drugs in forskolin-induced differentiated BeWo cells: alteration of gabapentin transport.

Previous studies have indicated that the expression levels of several transporters are altered during placental trophoblast differentiation. However, changes in the transport activities of therapeutic agents during differentiation must be comprehensively characterised. Antiepileptic drugs, including gabapentin (GBP), lamotrigine (LTG), topiramate, and levetiracetam, are increasingly prescribed during pregnancy. The objective of this study was to elucidate differences in the uptake of antiepileptic drugs during the differentiation process.Human placental choriocarcinoma BeWo cells were used as trophoblast models. For differentiation into syncytiotrophoblast-like cells, cells were treated with forskolin.The uptake of GBP and LTG was lower in differentiated BeWo cells than in undifferentiated cells. In particular, the maximum uptake rate of GBP transport was decreased in differentiated BeWo cells. Furthermore, GBP transport was trans-stimulated by the amino acids His and Met. We investigated the profiles of amino acids in undifferentiated and differentiated BeWo cells. Supplementation with His and Met, which demonstrated trans-stimulatory effects on GBP uptake, restored GBP uptake in differentiated cells. The findings of this study suggest that drug transport in BeWo cells can be altered before and after differentiation, and that the altered GBP uptake could be mediated by the intracellular amino acid status.

Evaluation of the strategies to reduce third-generation oral cephalosporins in dentistry at a Japanese academic hospital: An interrupted time series analysis.

WHAT IS KNOWN AND OBJECTIVE: Third-generation oral cephalosporins, especially cefcapene-pivoxil (CFPN-PI), have been used frequently in the Japanese dental field. In December 2014 and April 2016, the newly published clinical guidelines recommended the use of amoxicillin (AMPC). Thus, it is important to evaluate the impact of these guidelines on the prescription profiles of prophylactic antibiotics, clinical outcomes and cost-effectiveness of antibiotics. METHODS: We conducted a retrospective study to analyse an interrupted time series analysis from April 2013 to March 2020 at the Department of Dentistry of Hokkaido University Hospital. A segmented regression model was used to estimate the changes in the incidence of infectious complications following tooth extraction. Prescribed antibiotic data were evaluated via days of therapy (DOT). Antibiotic costs were calculated in terms of the Japanese yen (JPY). RESULTS AND DISCUSSION: We identified 17,825 eligible patients. The incidence rates of infectious complications (SSI + dry socket) and SSI after tooth extraction were 3.2% and 2.2%, respectively, during the entire period. The extraction of impacted third molars corresponded to 5.0% and 3.4%, respectively. However, their incidence rates were not significantly different during this period. The use of prophylactic antibiotics and antibiotic cost showed consistent trends following the implementation of guidelines. The mean DOT of CFPN-PI decreased (ranging from 4893.6 DOTs/1000 patients [March 2013 to November 2014] to 3856.4 DOTs/1000 patients [December 2014 to March 2016]; p < 0.001, and from 3856.4 DOTs/1000 patients [December 2014 to March 2016] to 2293.9 DOTs/1000 patients [April 2016 to March 2020]; p < 0.001). In contrast, the mean DOT of AMPC was found to be increased (ranging from 1379.7 DOTs/1000 patients [March 2013 to November 2014] to 3236.3 DOTs/1000 patients [December 2014 to March 2016]; p < 0.001, and from 3236.3 DOTs/1000 patients [December 2014 to March 2016] to 4597.8 DOTs/1000 patients [April 2016 to March 2020]; p < 0.001). The mean monthly cost was decreased (ranging from 905.3 JPY [March 2013 to November 2014] to 788.7 JPY [December 2014 to March 2016]; p = 0.003, and from 788.7 JPY [December 2014 to March 2016] to 614.0 JPY [April 2016 to March 2020]; p < 0.001). WHAT IS NEW AND CONCLUSION: After December 2014, prophylactic antibiotics were switched from CFPN-PI to AMPC, and the incidence rate of infectious complications was not significantly different over time. However, changing antibiotics is useful from a cost-effectiveness perspective.

Involvement of SLC16A1/MCT1 and SLC16A3/MCT4 in l-lactate transport in the hepatocellular carcinoma cell line.

Fourteen isoforms of the monocarboxylate transporter (MCT) have been reported. Among the MCT isoforms, MCT1, MCT2, and MCT4 play a role in l-lactate/proton cotransport and are involved in the balance of intracellular energy and pH. Therefore, MCT1, MCT2, and MCT4 are associated with energy metabolism processes in normal and pathological cells. In the present study, we evaluated the expression of MCT1, MCT2, and MCT4 and the contribution of these three MCT isoforms to l-lactate uptake in hepatocellular carcinoma (HCC) cells. In HepG2 and Huh-7 cells, l-lactate transport was pH-dependent, which is characteristic of MCT1, MCT2, and MCT4. Furthermore, l-lactate uptake was selectively inhibited by MCT1 and MCT4 inhibitors in HepG2 and Huh-7 cells. Kinetic analysis of HepG2 cells demonstrated that l-lactate uptake was biphasic. Although the knockdown of MCT1 and MCT4 in the HepG2 cells decreased the uptake of l-lactate, the knockdown of MCT2 had no effect on the uptake of l-lactate. Consequently, we concluded that both MCT1 and MCT4 were involved in the transport of l-lactate in HepG2 and Huh-7 cells at pH 6.0. In contrast, PXB-cells, freshly isolated hepatocytes from humanized mouse livers, showed lower MCT4 expression and l-lactate uptake at pH 6.0 compared to that in HCC cell lines. In conclusion, MCT4, which contributes to l-lactate transport in HCC cells, is significantly different in HCC compared to normal hepatocytes, and has potential as a target for HCC treatment.

Characterization of deoxyribonucleoside transport mediated by concentrative nucleoside transporters.

Human concentrative nucleoside transporters (CNTs) are responsible for cellular uptake of ribonucleosides; however, although it is important to better characterize CNT-subtype specificity to understand the systemic disposition of deoxyribonucleosides (dNs) and their analogs, the involvement of CNTs in transporting dNs is not fully understood. In this study, using COS-7 cells that transiently expressed CNT1, CNT2, or CNT3, we investigated if CNTs could transport not only ribonucleosides but also dNs, i.e., 2'-deoxyadenosine (dAdo), 2'-deoxyguanosine (dGuo), and 2'-deoxycytidine (dCyd). The cellular uptake study demonstrated that dAdo and dGuo were taken up by CNT2 but not by CNT1. Although dCyd was taken up by CNT1, no significant uptake was detected in COS-7 cells expressing CNT2. Similarly, these dNs were transported by CNT3. The apparent Km values of their uptake were as follows: CNT1, Km ＝ 141 µM for dCyd; CNT2, Km ＝ 62.4 µM and 54.9 µM for dAdo and dGuo, respectively; CNT3, Km ＝ 14.7 µM and 34.4 µM for dGuo and dCyd, respectively. These results demonstrate that CNTs contribute not only to ribonucleoside transport but also to the transport of dNs. Moreover, our data indicated that CNT1 and CNT2 selectively transported pyrimidine and purine dNs, respectively, and CNT3 was shown to transport both pyrimidine and purine dNs.

Preexisting autoimmune disease is a risk factor for immune-related adverse events: a meta-analysis.

PURPOSE: Patients with preexisting autoimmune disease (PAD) are often excluded from clinical trials assessing immune checkpoint inhibitors (ICIs). Therefore, the safety of ICI therapy in patients with PAD remains unclear. Herein, we evaluated the incidence of immune-related adverse events (irAEs) in patients with PAD when compared with non-PAD patients. METHODS: We searched MEDLINE/PubMed, Web of Science, and Google Scholar for eligible studies from inception to January 2021. Observational studies reporting the incidence of irAEs in patients with and without PAD were included. We then performed a meta-analysis of eligible studies using forest plots. The primary endpoint of this study was the incidence rate of irAEs between patients with and without PAD. RESULTS: We identified three prospective and three retrospective studies involving 206 patients with PAD and 3078 patients without PAD. In the meta-analysis, 128 patients with PAD (62.1%) experienced irAEs, which occurred in 51.9% of non-PAD patients, resulting in an odds ratio (OR) of 2.14 (95% confidence interval [CI] 1.58-2.89). In the subgroup analysis, the incidence of irAEs was significantly higher in patients with PAD (OR = 2.19, 95% CI [1.55-3.08]). Furthermore, no significant heterogeneity or publication bias was detected, indicating that our meta-analysis could be generalized to clinical settings. CONCLUSION: This meta-analysis demonstrated that PAD was a risk factor for irAE incidence. These results suggest that monitoring the occurrence of irAEs in patients with PAD is required to manage irAEs appropriately.

Pharmacological Inhibition of MCT4 Reduces 4-Hydroxytamoxifen Sensitivity by Increasing HIF-1α Protein Expression in ER-Positive MCF-7 Breast Cancer Cells.

The rate of glycolysis in cancer cells is higher than that of normal cells owing to high energy demands, which results in the production of excess lactate. Monocarboxylate transporters (MCTs), especially MCT1 and MCT4, play a critical role in maintaining an appropriate pH environment through lactate transport, and their high expression is associated with poor prognosis in breast cancer. Thus, we hypothesized that inhibition of MCTs is a promising therapeutic target for adjuvant breast cancer treatment. We investigated the effect of MCT inhibition in combination with 4-hydroxytamoxifen (4-OHT), an active metabolite of tamoxifen, using two estrogen receptor (ER)-positive breast cancer cell lines, MCF-7 and T47D. Lactate transport was investigated in cellular uptake studies. The cytotoxicity of 4-OHT was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In both cell lines evaluated, MCT1 and MCT4 were constitutively expressed at the mRNA and protein levels. [14C]-L-lactate uptake by both cells was significantly inhibited by bindarit, a selective MCT4 inhibitor, but weakly affected by 5-oxoploline (5-OP), a selective MCT1 inhibitor. The results of the MTT assay showed that combination with bindarit, but not 5-OP, decreased 4-OHT sensitivity. Bindarit significantly increased the levels of hypoxia-inducible factor-1α (HIF-1α) in MCF-7 cells. Moreover, HIF-1α knockdown significantly increased 4-OHT sensitivity, whereas induction of HIF-1α by hypoxia decreased 4-OHT sensitivity in MCF-7 cells. In conclusion, pharmacological MCT4 inhibition confers resistance to 4-OHT rather than sensitivity, by increasing HIF-1α protein levels. In addition, HIF-1α inhibition represents a potential therapeutic strategy for enhancing 4-OHT sensitivity.

Analysis of α-Defensin 5 Secretion in Differentiated Caco-2 Cells: Comparison of Cell Bank Origin.

α-Defensin 5 has a particularly broad antibacterial spectrum; it eliminates pathogenic microorganisms and regulates intestinal flora. Although Caco-2 cells are similar to small intestinal cells, it is unclear whether they secrete α-defensin 5. Therefore, we investigated whether Caco-2 cells secrete α-defensin 5 and determined the secretion mechanism using cells from three cell banks (ATCC, DSMZ, and RIKEN). The Caco-2 cell proliferation rate increased with the number of culture days, irrespective of cell bank origin. On the other hand, the alkaline phosphatase activity, which affects cell differentiation and the mRNA levels of several cytokines, such as interleukin 8 (IL-8), IL-6, IL-1ß, tumor necrosis factor-α (TNF-α), and IL-2, in the Caco-2 cells fluctuated with the number of culture days, and differed for each cell bank. α-Defensin 5 secretion was detected in all three cell bank Caco-2 cells; particularly, the ATCC Caco-2 cells grew linearly depending on the cell culture day as well as the levels of IL-8 and TNF-α mRNA. This suggested that α-defensin 5 secretion in the ATCC Caco-2 cells was associated with fluctuations in the mRNA levels of various cytokines, such as IL-8 and TNF-α. In conclusion, Caco-2 cells may be a simple model for screening health food components and drugs that affect α-defensin 5 secretion.

In vitro and in vivo evaluation of organic anion-transporting polypeptide 2B1-mediated pharmacokinetic interactions by apple polyphenols.

Organic anion-transporting polypeptide (OATP) 2B1 plays a critical role in the intestinal absorption of substrate drugs. Apple juice reportedly interacts with OATP2B1 substrate drugs. The purpose of this study was to investigate the effect of two apple polyphenols, phloretin and phloridzin, on OATP2B1-mediated substrate transport in vitro and to evaluate the effect of phloretin on rosuvastatin pharmacokinetics in rats.In vitro studies revealed that both polyphenols inhibited OATP2B1-mediated uptake of estrone-3-sulfate. Despite preincubation with phloretin and subsequent washing, the inhibitory effect was retained. Phloretin markedly decreased OATP2B1-mediated rosuvastatin uptake, with an IC50 value of 3.6 µM.On coadministering rosuvastatin and phloretin in rats, the plasma concentration of rosuvastatin 10 min after oral administration was significantly lower than that in the vehicle group. The area under the plasma concentration-time curve of rosuvastatin was not significant, showing a tendency to decrease in the phloretin group when compared with the vehicle group. The in-situ rat intestinal loop study revealed the inhibitory effect of phloretin on rosuvastatin absorption.Phloretin has potent and long-lasting inhibitory effects on OATP2B1 in vitro. Phloretin may inhibit OATP2B1-mediated intestinal absorption of rosuvastatin; however, it failed to significantly impact the systemic exposure of rosuvastatin in rats.

Kinetic analysis of cystine uptake and inhibition pattern of sulfasalazine in A549 cells.

Cystine/glutamate transporter (xCT) is an antiporter involved in cystine uptake and glutamate efflux. However, there are very few reports regarding the kinetic analysis of xCT for cystine uptake using cancer cell lines, as well as the inhibition pattern of sulfasalazine, an inhibitor of xCT, for cystine uptake. Therefore, the purpose of this study was to clarify the kinetics of xCT in A549 cells, human lung cancer cells, and to reveal the inhibition pattern of sulfasalazine. Cystine uptake occurred in a time-dependent manner, with linear cystine uptake observed for 5 min. Additionally, sulfasalazine inhibited cystine uptake in a concentration-dependent manner, presenting an IC50 value of 24.7 ± 5.6 µM. Cystine uptake was saturated with increasing concentration, demonstrating Km and Vmax values of 179.4 ± 26.7 µM and 30.4 ± 2.3 nmol/min/mg protein, respectively. Moreover, during cystine uptake with sulfasalazine, Km and Vmax were >300 µM and 8.0 ± 1.5 nmol/min/mg protein, respectively, suggesting that sulfasalazine might demonstrate a mixed inhibition pattern. Furthermore, xCT siRNA decreased the xCT mRNA level and reduced cystine uptake. In conclusion, xCT was involved in the cystine uptake in A549 cells and sulfasalazine showed a mixed inhibition pattern to xCT.

Identification of the essential extracellular aspartic acids conserved in human monocarboxylate transporters 1, 2, and 4.

Human monocarboxylate transporters (hMCTs) 1-4 transport monocarboxylates, such as l-lactate and pyruvate, as well as H+ across the plasma membrane. hMCT1, 2, and 4 play important roles in energy balance, pH homeostasis. However, the molecular mechanism of these transporters, especially their pH dependency, remains unknown. The aim of this study was to identify the residues involved in the pH dependence of hMCT1, 2, and 4. Firstly, we focused on the effects of extracellular acids of hMCT1. l-Lactate uptake assay and site-directed mutagenesis revealed that the aspartic acid of hMCT1 (hMCT1 D414) was an important residue conserved in MCT1, 2, and 4 (hMCT2 D398 and hMCT4 D379). Because the functional characteristic of hMCT2-mediated l-lactate transport has not been reported, we built a hMCT2-expressing system using Xenopus laevis oocytes. The transport activity of hMCT2 was enhanced by co-expression with embigin, an ancillary protein, and kinetic analysis of hMCT2-mediated l-lactate uptake revealed that the apparent Km value (0.32 ± 0.02 mM) was lower than that mediated by hMCT1 and 4. Finally, we investigated the conserved aspartic acids of hMCT2 and 4, and revealed that these residues were essential for l-lactate transport. These findings suggested that the extracellular aspartic acids conserved in hMCT1, 2, and 4 played important roles in transport activity and pH dependency, and can function as a first step of substrate and H+ recognition and transport from the extracellular to the intracellular region. These findings contributed to enhance our understanding of the transport process of hMCT1, 2, and 4.

Different mechanisms of cisplatin resistance development in human lung cancer cells.

Cisplatin (CDDP) is a highly potent and important anticancer drug in lung cancer treatment. Long-term use of an anticancer agent causes resistance in cancer cells, and CDDP resistance involves multiple mechanisms. As the mechanism of resistance development differs depending on the cancer cell types, we aimed to evaluate the detailed mechanism of resistance to CDDP in two types of lung cancer cells: SBC-3 and A549 cells. The CDDP-resistant SBC-3/DDP and A549/DDP cells were established through continuous treatment with a gradually increasing dose of CDDP. The viability of SBC-3/DDP and A549/DDP cells treated with CDDP was 3.68 and 2.08 times higher than that of the respective parental cells. Moreover, SBC-3/DDP cells showed significantly increased cystine/glutamate transporter (xCT) mRNA level, and A549/DDP cells showed markedly increased sex determining region Y-box 2 (SOX2) mRNA level. Moreover, the uptake of cystine, a substrate of xCT, was higher in SBC-3/DDP cells than in SBC-3 cells, and cystine uptake in A549/DDP cells was not different from that in A549 cells. In addition, co-treatment with CDDP and sulfasalazine, an xCT inhibitor, showed lower the concentration of 50% inhibition for cell viability than CDDP alone in SBC-3 and SBC-3/DDP cells, but not in A549 and A549/DDP cells. Furthermore, SBC-3 cells transiently overexpressing xCT were resistant to CDDP, and xCT knockdown in A549/DDP cells did not significantly change the level of SOX2 mRNA and viability of cells upon CDDP treatment. In conclusion, the two lung cancer cell lines showed different mechanisms of resistance to CDDP.

Molecular characterization of the orphan transporter SLC16A9, an extracellular pH- and Na+-sensitive creatine transporter.

Human monocarboxylate transporters (hMCTs) mediate the transport of monocarboxylates across plasma membranes. One such transporter, hMCT9, has been shown to be related to serum uric acid levels and the risk of renal overload gout. However, the functional characteristics of hMCT9 remain unknown. The aim of this study was to investigate the expression and localization of hMCT9 using a Xenopus laevis oocyte heterologous expression system and characterize its transport properties. Kinetic analysis of hMCT9-mediated creatine uptake revealed that uptake consisted of two components, with apparent Km values of 237 mm (low-affinity) and 23.7 mm (high-affinity), respectively. The transport activity of hMCT9 was dependent on the extracellular pH and activity sharply increased with increasing pH. Under Na+-free conditions, hMCT9-mediated creatine uptake was reduced by one-half, indicating that hMCT9 is a Na+-sensitive transporter. Moreover, carbonyl cyanide 3-chlorophenylhydrazone (a protonophore) inhibited hMCT9 activity, whereas valinomycin (a K+-ionophore) did not inhibit the transporter. These results suggest that hMCT9 is susceptible to changes in H+ gradients. A cis-inhibition assay of hMCT9-and hMCT12-mediated creatine transport revealed that cyclocreatine, creatine, guanidineacetate, and 3-guanidinopropionate are recognized by the transporter, and 4-guanidinobutyrate and guanidinoethyl sulfonate selectively inhibited hMCT9 activity. These findings demonstrate that hMCT9 is an extracellular pH- and Na+-sensitive creatine transporter.

Association Between N-Desmethylclozapine and Clozapine-Induced Sialorrhea: Involvement of Increased Nocturnal Salivary Secretion via Muscarinic Receptors by N-Desmethylclozapine.

Clozapine-induced sialorrhea (CIS) is a common side effect of clozapine. There is no established standard treatment of CIS since the underlying mechanism remains unknown. This study aimed to elucidate the mechanisms involved in CIS. In our clinical study, a prospective observational study evaluated the association between serum and saliva concentrations of clozapine or its metabolites and Drooling Severity and Frequency Scale (DSFS) score. In our in vivo study, we first developed a new CIS animal model; subsequently, we measured salivary secretion and concentrations of clozapine or its metabolites in the animal model. In our in vitro study, we measured the calcium ion (Ca2+) response to evaluate the effect of clozapine or its metabolites on human salivary gland cell line (HSY cells) and then examined whether their effect was inhibited by atropine. In our clinical study, serum and saliva N-desmethylclozapine concentrations were significantly correlated with nocturnal DSFS score. In our in vivo study, daily single oral administration of 100 mg/kg clozapine for 7 days significantly increased salivary secretion in rats. Furthermore, N-desmethylclozapine concentrations in serum and submandibular glands of the rats were higher than clozapine concentrations. In our in vitro study, N-desmethylclozapine only elicited an increase in the intracellular Ca2+ in HSY cells. N-desmethylclozapine-induced Ca2+ responses were inhibited by atropine. These results suggest that N-desmethylclozapine is implicated in CIS by increasing nocturnal salivation via the muscarinic receptors. Moreover, our developed animal model that reflects CIS in clinical condition plays a key role as a bridge between basic and clinical research. SIGNIFICANCE STATEMENT: Clozapine-induced sialorrhea (CIS) is a severe and frequent adverse reaction, but the mechanism underlying CIS is less well understood. This paper reports that N-desmethylclozapine, a metabolite of clozapine, is implicated in CIS by increasing nocturnal salivation via the muscarinic receptors and that oral administration of clozapine at 100 mg/kg once daily for 7 days to rat is the optimum method for establishing the new animal model reflecting the clinical scenario of CIS.

Homology modeling and site-directed mutagenesis identify amino acid residues underlying the substrate selection mechanism of human monocarboxylate transporters 1 (hMCT1) and 4 (hMCT4).

Human monocarboxylate transporters (hMCTs/SLC16As) mediate the transport of monocarboxylic compounds across plasma membranes. Among the hMCTs, hMCT1 and hMCT4 are expressed in various tissues, and transport substrates involved in energy metabolism. Both transporters mediate L-lactate transport, but, although hMCT1 also transports L-5-oxoproline (L-OPro), this compound is minimally transported by hMCT4. Thus, we were interested in the molecular mechanism responsible for the difference in substrate specificity between hMCT1 and hMCT4. Therefore, we generated 3D structure models of hMCT1 and hMCT4 to identify amino acid residues involved in the substrate specificity of these transporters. We found that the substrate specificity of hMCT1 was regulated by residues involved in turnover number (M69) and substrate affinity (F367), and these residues were responsible for recognizing (directly or indirectly) the -NH- moiety of L-OPro. Furthermore, our homology model of hMCT1 predicted that M69 and F367 participate in hydrophobic interactions with another region of hMCT1, emphasizing its potentially important role in the binding and translocation cycle of L-OPro. Mutagenesis experiments supported this model, showing that efficient L-OPro transport required a hydrophobic, long linear structure at position 69 and a hydrophobic, γ-branched structure at position 367. Our work demonstrated that the amino acid residues, M69 and F367, are key molecular elements for the transport of L-OPro by hMCT1. These two residues may be involved in substrate recognition and/or substrate-induced conformational changes.