BackgroundIn the central nervous system, astrocytic L-glutamate (L-Glu) transporters maintain extracellular L-Glu below neurotoxic levels, but their function is impaired with neuroinflammation. Microglia become activated with inflammation; however, the correlation between activated microglia and the impairment of L-Glu transporters is unknown.MethodsWe used a mixed culture composed of astrocytes, microglia, and neurons. To quantify L-Glu transporter function, we measured the extracellular L-Glu that remained 30 min after an application of L-Glu to the medium (the starting concentration was 100 μM). We determined the optimal conditions of lipopolysaccharide (LPS) treatment to establish an inflammation model without cell death. We examined the predominant subtypes of L-Glu transporters and the changes in the expression levels of these transporters in this inflammation model. We then investigated the role of activated microglia in the changes in L-Glu transporter expression and the underlying mechanisms in this inflammation model.ResultsBecause LPS (10 ng/mL, 72 h) caused a significant increase in the levels of L-Glu remaining but did not affect cell viability, we adopted this condition for our inflammation model without cell death. GLAST was the predominant L-Glu transporter subtype, and its expression decreased in this inflammation model. As a result of their release of L-Glu, activated microglia were shown to be essential for the significant decrease in L-Glu uptake. The serial application of L-Glu caused a significant decrease in L-Glu uptake and GLAST expression in the astrocyte culture. The hemichannel inhibitor carbenoxolone (CBX) inhibited L-Glu release from activated microglia and ameliorated the decrease in GLAST expression in the inflammation model. In addition, the elevation of the astrocytic intracellular L-Glu itself caused the downregulation of GLAST.ConclusionsOur findings suggest that activated microglia trigger the elevation of extracellular L-Glu through their own release of L-Glu, and astrocyte L-Glu transporters are downregulated as a result of the elevation of astrocytic intracellular L-Glu levels, causing a further increase of extracellular L-Glu. Our data suggest the new hypothesis that activated microglia collude with astrocytes to cause the elevation of extracellular L-Glu in the early stages of neuroinflammation.

Since gonadal female hormones act on and protect neurons, it is well known that the female brain is less vulnerable to stroke or other brain insults than the male brain. Although glial functions have been shown to affect the vulnerability of the brain, little is known if such a sex difference exists in glia, much less the mechanism that might cause gender-dependent differences in glial functions. In this study, we show that in vitro astrocytes obtained from either female or male pups show a gonadal hormone-independent phenotype that could explain the gender-dependent vulnerability of the brain. Female spinal astrocytes cleared more glutamate by GLAST than male ones. In addition, motoneurons seeded on female spinal astrocytes were less vulnerable to glutamate than those seeded on male ones. It is suggested that female astrocytes uptake more glutamate and reveal a stronger neuroprotective effect against glutamate than male ones. It should be noted that such an effect was independent of gonadal female hormones, suggesting that astrocytes have cell-autonomous regulatory mechanisms by which they transform themselves into appropriate phenotypes.

Aprepitant is a known inducer of CYP2C9, the main warfarin-metabolizing enzyme. Consequently, coadministration of these two drugs may result in reduction of the anticoagulation activity of warfarin. However, the nature and degree of time-dependent changes in prothrombin time international normalized ratio (PT-INR) after aprepitant and warfarin co-treatment in patients receiving anticancer chemotherapy has not been elucidated. We retrospectively examined the changes in warfarin dose, PT-INR, and warfarin sensitivity index (WSI; average of PT-INR value/average of daily warfarin dose) during four weeks, i.e., one week before and three weeks after aprepitant administration. The mean and standard deviation values of WSI for one week before and one, two, and three weeks after the beginning of aprepitant administration were 0. Aprepitant has been reported to be a moderate inhibitor and inducer of CYP3A4, as well as an inducer of CYP2C9.2,3) The administration of aprepitant (125 mg) is recommended one hour prior to initiating chemotherapy treatment, followed by doses of 80 mg in the morning of the second and third days.Warfarin is the most widely prescribed anticoagulant drug, and has been used for the treatment and prevention of thromboembolic diseases.4) Drug interactions with this oral anticoagulant are clinically relevant since an interaction leading to enhanced action may be associated with an increased risk of hemorrhage. Conversely, interactions that decrease warfarin plasma concentrations might decrease the anticoagulant effect of warfarin. Warfarin is clinically administered as a racemic mixture of R-and S-warfarin, which differ in the potency of their anticoagulation effect, with the potency of S-warfarin being three to five times higher than that of R-warfarin.5) The R-and S-forms also have different metabolic pathways: while R-warfarin is metabolized by CYP3A4 and CYP1A2, S-warfarin is mainly metabolized by CYP2C9. 6)The manufacturer cautions that co-administration of aprepitant with warfarin may result in a clinically significant decrease in prothrombin time international normalized ratio (PT-INR) owing to the induction of CYP2C9 by aprepitant, which causes S-warfarin concentration to decrease. Compared to placebo-treated subjects, in healthy subjects stabilized on warfarin and administered a 3-d regimen of aprepitant, S-warfarin concentration and PT-INR were found to decrease, respectively, by 34 and 14% on day 8 after aprepitant administration.2) It is clinically important to elucidate whether aprepitant causes a reduction of the anticoagulation activity of warfarin in patients receiving anticancer chemotherapy, as well as in healthy subjects. We previously reported two cases in which treatment with aprepitant persistently altered antithrombotic control in patients receiving warfarin. 7) However, the nature and degree of time-dependent changes in PT-INR after aprepitant and anticancer co-treatment remains unknown. In the present study, we aimed to clarify the effects of a drug-drug interaction between apr...