Takao Shimazoe

Estrogen differentially regulates expression of Per1 and Per2 genes between central and peripheral clocks and between reproductive and nonreproductive tissues in female rats

Although it has long been established that estrogen alters circadian rhythms in behavior, physiology, and reproductive functions in mammals, the molecular mechanism for these effects remains unknown. To explore the possibility that estrogen affects circadian rhythms by changing the expression of clock‐related genes, we investigated the effects of chronic treatment with 17β‐estradiol (E2) on the expression of Per1 and Per2 genes in the brain (suprachiasmatic nucleus and cerebral cortex) and periphery (liver, kidney, and uterus) of ovariectomized rats by means of in situ hybridization and northern blotting. In the brain, E2 treatment advanced the peak of Per2 mRNA expression in the SCN; however, it failed to affect the rhythm of Per2 mRNA expression in the CX and Per1 mRNA expression in both the SCN and the CX. In nonreproductive peripheral tissues (liver and kidney), E2 delayed the phase and increased the amplitude of Per1 mRNA expression. In the reproductive tissues (uterus), biphasic rhythms in Per1 and Per2 mRNA were observed after E2 treatment. These findings suggest that the effects of estrogen are different between central and peripheral clock in the brain, and between reproductive and nonreproductive tissues in the periphery.

Effects of 16-Week Consumption of Caffeinated and Decaffeinated Instant Coffee on Glucose Metabolism in a Randomized Controlled Trial

Objective. Observational studies have shown a protective association between coffee consumption and type 2 diabetes mellitus whereas caffeine or caffeinated coffee acutely deteriorates glucose tolerance. We investigated the effects of chronic drinking of instant coffee on glucose and insulin concentrations during a 75 g oral glucose tolerance test. Methods. Overweight men with a mild-to-moderate elevation of fasting plasma glucose were randomly allocated to a 16-week intervention of consuming 5 cups of caffeinated (n = 17) or decaffeinated (n = 15) instant coffee per day or no coffee (n = 13). Results. The caffeinated coffee group showed statistically significant decreases in the 2-hour concentrations and the area under the curve of glucose while neither decaffeinated coffee nor coffee group showed such a change. Waist circumstance decreased in the caffeinated coffee group, increased in the decaffeinated coffee group, and did not change in the noncoffee group (P = 0.002). With adjustment for the change in waist circumference, caffeinated and decaffeinated coffee consumption were associated with a modest decrease in the postload glucose levels. Conclusion. Both caffeinated and decaffeinated coffee may be protective against deterioration of glucose tolerance.

Antitumor Effect of DX‐8951, a Novel Camptothecin Analog, on Human Pancreatic Tumor Cells and Their CPT‐11‐resistant Variants Cultured in vitro and Xenografted into Nude Mice

DX‐8951 is a novel water‐soluble derivative of camptothecin. We evaluated the effects of DX‐8951 on the growth of several pancreatic tumor cell lines in vitro and in vivo. In vitro cytotoxic activity of DX‐8951 against SUIT‐2 and KP‐1N cells, as indicated by IC50 value, was several times more potent than that of SN‐38, an active metabolite of CPT‐11, and dozens of times more potent than that of SK&F104864 (topotecan). DX‐8951 also showed the greatest cytotoxicity against CPT‐11‐resis‐tant variants, SUIT‐2/CPT‐11 and KP‐1N/CPT‐11 cells, and the cross‐resistance of these cells to DX‐8951 was lower than that to SN‐38 and SK&F104864. Topoisomerase 1 inhibitory activity of DX‐8951 was about three‐fold stronger than that of SN‐38, as measured in crude nuclear extract obtained from SUIT‐2 cells. DX‐8951 induced DNA fragmentation, a specific feature of apoptosis, in SUIT‐2 cells more effectively than SN‐38. DX‐8951 exhibited potent antitumor effects against SUIT‐2 in a solid tumor model and in a liver metastasis model, in which tumor cells were xenografted sub‐cutaneously and intrasplenically, respectively, into nude mice. The in vivo effects were closely similar to or somewhat superior to those of CPT‐11, DX‐8951 also showed significant antitumor effects against SUIT‐2/CPT‐11 solid tumors, against which CPT‐11 had no effect. These results suggest that, on the basis of its strong antitumor activity and effectiveness against CPT‐11‐resistant tumors, DX‐8951 may be a useful therapeutic agent in the treatment of human cancer. The potent cytotoxicity of DX‐8951 may result from strong inhibition of topoisomerase I, which may then trigger apoptotic cell death.

Enhancement of dopamine release from the striatum through metabotropic glutamate receptor activation in methamphetamine sensitized rats

An intracerebral microdialysis technique was applied to study the effect of metabotropic glutamate receptor (mGluR) agonist on dopamine release in the striatum of methamphetamine (MAP)-sensitized rats. Rats were treated with MAP (1 mg/kg, i.p.) once daily for 6 consecutive days, followed by a 6-day withdrawal. Perfusion of 0.1 mM (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid through a microdialysis probe placed in the striatum enhanced the extracellular dopamine level, and induced stereotyped behavior in MAP-sensitized rats. The enhancement of dopamine release and the stereotyped behavior were attenuated by co-perfusion of 0.4 mM RS-alpha-methyl-4-carboxyphenyl-glycine, a mGluR antagonist. The present results suggest that mGluRs may be involved in the expression of MAP-induced sensitization.

Expression of long-term potentiation of the striatum in methamphetamine- sensitized rats

We examined the change of corticostriatal glutamatergic neuronal transmission in striatal slices of methamphetamine (MAP)-sensitized rats in vitro. Tetanic stimulation induced long-term depression (LTD) of the field potential in the striatum of saline-treated rats. However, it induced long-term potentiation (LTP) in the striatum of MAP-sensitized rats. This LTP was significantly suppressed by a N-methyl-D-aspartate (NMDA) receptor antagonist, aminophosphonovaleric acid (APV). These results suggest that LTP is expressed in the striatum of MAP-sensitized rats, and that NMDA receptors are indispensable for the LTP formation.

Reversal of methamphetamine-induced behavioral sensitization by repeated administration of a dopamine D1 receptor agonist

Repeated intermittent administration of methamphetamine (MAP) produces an enduring hypersensitivity to the motor stimulant effect of MAP, termed behavioral sensitization. Dopamine plays a critical role in the development and expression of behavioral sensitization. Here, we investigated whether a dopamine D1 receptor agonist could reverse behavioral sensitization to MAP. Administration of MAP (1.0 mg/kg, i.p.) to rats once every 3 days for a total of 5 times (days 1-13) induced the enhancement of locomotor activity after MAP challenge (0.5 mg/kg, i.p.) on day 20, verifying the development of behavioral sensitization. The MAP-sensitized rats then received a dopamine D1 agonist, R-(+)-SKF38393 (3.0 mg/kg, i.p.), once a day for 7 consecutive days (days 21-27). Behavioral analysis on days 30 and 41 revealed that the enhanced locomotor activity was reversed by repeated R-(+)-SKF38393 administration. Moreover, repeated R-(+)-SKF38393 administration reversed the increased dopamine release in the striatum after MAP challenge on day 41. Thus, repeated administration of the dopamine D1 receptor agonist induces the reversal of established behavioral sensitization to MAP and of increased dopamine release in the striatum, lasting for at least 2 weeks. Dopamine D1 receptor agonists may be useful therapeutic agents for the treatment of psychostimulant addiction.

Maternal Exposure to Dioxin Disrupts Gonadotropin Production in Fetal Rats and Imprints Defects in Sexual Behavior

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are a class of environmental pollutants with suspected toxic effects on reproductive and developmental processes. This study investigated a hypothesis that maternal exposure to TCDD damages gonadotropin-regulated steroidogenesis in fetal gonads to imprint defects in sexual behavior as well as the maturation of gonadal tissues. Oral administration of 1 μg/kg TCDD to pregnant Wistar rats at gestational day (GD) 15 attenuated the expression of luteinizing hormone (LH), a regulator of gonadal steroidogenesis, in the pituitaries of male and female fetuses at GD20. TCDD treatment also reduced the fetal expression of testicular and ovarian steroidogenic proteins, including steroidogenic acute-regulatory protein. These changes in pituitary and gonadal proteins were fetus-specific, and this seems not to be because of the greater delivery of TCDD to the brain of fetuses than adults. This is because a reduction in LH production was not reproduced even although TCDD was administered intraventricularly to adult rats. Direct supplementation of equine chorionic gonadotropin (eCG), an LH-mimicking hormone, to TCDD-exposed fetuses at GD17 restored the reduced expression of gonadal steroidogenic proteins. Maternal exposure to TCDD delayed the development of gonadal tissues in male and female pups and impaired their sexual behavior. However, eCG treatment at the fetal stage again restored not only tissue maturation but also many of the behavioral defects that occurred at adulthood. These results demonstrate that TCDD disrupts steroidogenesis in fetuses by targeting pituitary gonadotropin production and imprints demasculinization in males and defeminization in females in terms of their copulatory behavior.

EXPRESSION OF N-METHYL-d-ASPARTATE RECEPTOR- DEPENDENT LONG-TERM POTENTIATION IN THE NEOSTRIATAL NEURONS IN AN IN VITRO SLICE AFTER ETHANOL WITHDRAWAL OF THE RAT

To examine changes in corticostriatal synaptic transmission in rats with ethanol withdrawal syndrome, intracellular and extracellular responses to subcortical white matter stimulation were recorded in neostriatal slice preparations. The resting membrane potential, input resistance and depolarizing postsynaptic potentials to single cortical white matter stimulation were similar in the neostriatum of naive and ethanol withdrawal rats. Repetitive stimulation of the white matter induced more pronounced N-methyl-D-aspartate receptor-mediated postsynaptic potentials in ethanol withdrawal than naive rat neostriatum. In intracellular recording, tetanic stimulation (50 Hz, 20 s) induced more pronounced post-tetanic potentiation of depolarizing postsynaptic potentials in the neostriatum of ethanol withdrawal than naive rats. However, in extracellular recording, tetanic stimulation induced smaller post-tetanic depression of population spikes in the neostriatum of ethanol withdrawal than naive rats. Tetanic stimulation of the subcortical white matter induced long-term potentiation of postsynaptic potentials and population spikes in the ethanol withdrawal rat neostriatum, while long-term depression was evoked in the naive rat neostriatum. The induction of long-term potentiation was blocked by D-2-amino-5-phosphonovaleric acid or 7-chlorokynurenic acid, N-methyl-D-aspartate receptor antagonists, but not by (RS)-methyl-4-carboxyphenyl-glycine, a metabotropic glutamate receptor antagonist. Dopamine also significantly depressed the induction of long-term potentiation in ethanol withdrawal rat neostriatum and this depressant effect was antagonized by the D2 antagonist L-sulpiride but not by the D1 antagonist SCH23390. These results indicate that the N-methyl-D-aspartate component of the corticostriatal glutamatergic responses, which might be necessary for induction of long-term potentiation, was enhanced in ethanol withdrawal rats. The depression of long-term potentiation induction by activation of D2 receptor suggests that corticostriatal N-methyl-D-aspartate response or intracellular mechanisms involving in the induction of the long-term potentiation can be suppressed by D2 activation and that the D2 effects are inhibited in the neostriatum of ethanol withdrawal rats.

In vivo delivery of glial cell-derived neurotrophic factor across the blood-brain barrier by gene transfer into brain capillary endothelial cells

The gene encoding mouse glial cell-derived neurotrophic factor (mGDNF gene) was transfected into brain capillary endothelial cells (BCECs) with the aim of delivering the gene product extensively into the brain parenchyma by making use of the secretory function of BCECs. First, we transfected mGDNF gene into cultured BCECs (MBEC4; mouse brain capillary endothelial cells) in vitro. The amount of mGDNF protein secreted from the transfected cells into the medium was 1500 to 3200 pg/mg of cell protein per day, being about sevenfold higher than that accumulated intracellularly. Furthermore, the basolateral-directed secretion of mGDNF protein from the transfected MBEC4 cells was fivefold higher than the apical-directed secretion. Next, the hemagglutination virus of Japan (HVJ)-liposomes encapsulating mGDNF gene were administered to rats in vivo via the internal carotid artery. The transfected rats showed a marked increase in the brain level of GDNF as assessed by means of enzyme-linked immunosorbent assay (ELISA) and Western blotting on day 3 after the administration, and the level remained significantly elevated for at least 12 days. Furthermore, immunohistochemical staining revealed an increase in GDNF immunoreactivity throughout the transfected forebrain. These results indicate that the gene was successfully transferred in vivo from HVJ-liposomes into BCECs, where it was expressed, and the gene product was secreted into the brain. Then, using this delivery method, we evaluated the protective effect for dopamine neuron against a retrograde 6-hydroxydopamine (6-OHDA) lesion, as assessed by behavioral and neurochemical indices.

Neurotropin reverses paclitaxel-induced neuropathy without affecting anti-tumour efficacy

Paclitaxel is a commonly used anticancer drug, but it frequently causes peripheral neuropathy. Neurotropin, a non-protein extract from inflamed rabbit skin inoculated with vaccinia virus, has been used to treat various chronic painful conditions. In the present study, we investigated the effect of neurotropin on the paclitaxel-induced neuropathy in rats. Repeated administration of paclitaxel induced mechanical allodynia, cold hyperalgesia, and motor dysfunction. These neuropathies were mostly reversed by the repeated administration of neurotropin. Furthermore, neurotropin ameliorated the paclitaxel-induced axonal degeneration in cultured PC12 and rat dorsal root ganglion cells, and in rat sciatic nerve. In addition, neurotropin did not affect the microtubule aggregation or anti-tumour effect induced by paclitaxel in the tumour cell lines or tumour cells-implanted mice. These results suggest that neurotropin reverses the paclitaxel-induced neuropathy without affecting anti-tumour activity of paclitaxel, and therefore may be useful for the paclitaxel-induced neuropathy in clinical settings.