The ambient temperature (Ta) of the delivery room has been under discussion because high Ta is suspected to relate to neonatal encephalopathy. We investigated the effect of Ta on the voluntary motion ability of adult rat models after neonatal hypoxic-ischemic (HI) insult. Under inspired anesthesia, the left carotid artery of a 7-day-old rat was ligated followed by hypoxic insults made by delivering 8% oxygen at 40 °C for 15 min. The rats were returned to their mother for 1 hr, and then placed in a box at one of two Ta of 40 °C (n=5; HY group) and 34 °C (n=5; HI group). A sham group (n=5; S group) was also established. Three months later, the voluntary motion ability of rats was assessed using an activity wheel. The device was composed of a rotary cage for free running with an attached cage with free access to food and water with a 12:12 LD cycle. We measured the voluntary motion of rats for 72 consecutive hrs. Self-motion was assessed every 24 hrs from the number of rotations. After habituation to the device, we put a rat from one of the three groups in the cage. The S group rotated the wheel up to 1,200 from 500. The HY group increased the number of rotations during the first 48 hours and decreased afterwards. Although no significant difference was observed between rotation times within the HY group and the S group, rotation times in the HI group were significantly fewer than those in the HY group and S group. These results suggest that the HI group may not be accustomed to the sudden change of environment produced by the device. [J Physiol Sci. 2008;58 Suppl:S103]

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Capsaicin (CAP) and dihydrocapsaicin (DIH) are the main pungent components of capsaicinoids, which account for about 90% of the pungent ingredient in capsicum. These components are known to change immune responses by inducing adrenal hormones, neuropeptides and cytokines. However, the effects of capsaicinoids on the number of immune cells are still unknown. Therefore, the effects of CAP and DIH on the numbers of total white blood cells (WBCs), neutrophil, lymphocytes, monocyte, eosinophil and basophil were studied. The effects of DIH on the numbers of T-lymphocyte, B-lymphocyte and natural killer (NK) cells were also examined. Adult male SD rats were divided into three (CAP, DIH and the control (CON)) groups. CAP and DIH (each dose = 3.0 mg/kg BW) were administered via sc injection. The numbers of total WBCs in CAP and DIH were significantly higher than that in CON. The number of neutrophil in DIH was higher than those in CAP and CON. The numbers of lymphocytes in CAP and DIH were significantly lower than that in CON. The number of B-lymphocyte in DIH markedly decreased. However, the numbers of T-lymphocyte and NK cells were not changed. The numbers of monocyte, eosinophil and basophil showed no significant changes among three groups. In conclusion, the administration of CAP and DIH decreased the numbers of lymphocytes and B-lymphocyte, and increased the numbers of total WBCs and neutrophil without changing the numbers of monocyte, eosinophil and basophil. [J Physiol Sci. 2008;58 Suppl:S103]

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Dmbx1 is a paired-class homeodomain transcription factor of unknown function. To elucidate the physiological role, we generated mice deficient in Dmbx1 (Dmbx1^−/− mice). Dmbx1^−/− mice exhibit severe leanness associated with hypophagia and hyperactivity. To clarify the mechanism of their leanness, Dmbx1^−/− mice were crossed with lethal yellow (A^y/a) mice, in which the ectopic expression of agouti protein causes marked obesity and diabetes mellitus associated with hyperphagia and insulin resistance. Interestingly, overexpression of agouti in Dmbx1^−/− mice failed to induce the A^y/a phenotype. In Dmbx1^−/− mice, administration of AgRP did not increase cumulative food intake over 24 and 48 h. In addition, Dmbx1 was shown to be expressed at E15.5 in lateral parabrachial nucleus (LPB), rostral nucleus of the tractus solitarius (NTS), dorsal motor nucleus of the vagus (DMV), and reticular nucleus (RET) in the brain stem, all of which receive melanocortin signaling, indicating Dmbx1 participates in the development of the neural network for the signaling. Thus, Dmbx1 plays an important role in eliciting pleiotropic action of AgRP, regulating energy homeostasis and behavior. [J Physiol Sci. 2008;58 Suppl:S104]

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It is still unclear whether catechin influences the brain function. We studied the effects of catechin on serotonin (5-HT) release from hippocampus, since 5-HT release in the brain dominates in uncomfortable conditions. 5-HT was measured in the hippocampus of unconscious Wistar rat, using in vivo microdialysis technique. We used five catechin derivatives; (+)catechin (CAT), (-)epigallocatechin gallate (EGCG), (-)epigallocatechin (EGC), (-)epicatechin gallate (ECG), (-)epicatechin (EC). Catechin concentration used was 10μM, except for CAT (1μM–1mM), and these compounds containing in the Ringer solution were perfused in the brain for 100 min. The 5-HT release by catechin was also studied in the conditions of high K or low K, and the addition of tetrodotoxin (TTX, 1μM), monoamineoxidase (MAO) inhibitors (pargyline, 0.1mM; clorgyline, 1mM) , and SSRI (fluvoxamine, 1mM). All catechin derivatives remarkably increased 5-HT release. EC was the most effective (270 times) in increasing 5-HT release, followed by CAT, EGC, ECG, EGCG in that order. CAT increased the 5-HT release in a concentration dependent manner, indicating that the ED₅₀ is about 0.1mM. In addition, oral application of CAT (0.1mM) also led to increase of 5-HT release, suggesting that CAT penetrates the blood-brain barrier. 5-HT release by CAT was significantly reduced in the presence of MAO inhibitors and/or SSRI, while the release remained unchanged in the conditions of high K or low K, and the addition of TTX. These findings suggest that catechin functions in the brain as a MAO inhibitor and/or a SSRI. [J Physiol Sci. 2008;58 Suppl:S104]

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Recently, we demonstrated that zinc deficient rats enhanced their preference to high concentration of sodium chloride. In the present study, we conducted behavioral experiments to investigate whether or not calcium deficient rats (CaX) also enhanced their preference to sodium chloride. Results were as follows; in the long-term (48 h) two-bottle preference test, the preference percents for 0.3 and 1.0M NaCl in the CaX rats were higher than those in the control rats. But in the short-term (10 min) test, there was no significant difference in the preference percents between CaX and control rats. When the CaX rats transected the chorda tympani and glossopharyngeal nerves were used for long-term two-bottle preference test, there was no significant difference between the preference percents for 0.3 and 1.0M NaCl in the CaX and the sham rats. These results suggest that the enhancement of preference for sodium chloride is caused by the post-ingestive effect rather than taste effect. [J Physiol Sci. 2008;58 Suppl:S104]

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Leptin is a hormone that stimulates glucose uptake in some peripheral tissues and enhances whole-body insulin sensitivity through the sympathetic nervous system. However, the molecular mechanism remains to be elucidated. In the present study, we investigated how leptin and melanocortin receptor agonist (MT II) increase glucose uptake in peripheral tissues in mice. Microinjection of leptin into the ventromedial hypothalamus increased glucose uptake in skeletal muscle (SM) and brown adipose tissue (BAT). Intracerebroventricular injection of MT II also increased glucose uptake and serine phosphorylation of Akt in SM and BAT. When MT II was injected intracerebroventricularly and insulin is administered intravenously maintaining plasma glucose level, the glucose uptake by SM increased beyond the value in response to insulin stimulation alone. In mouse myoblastoma C2C12 cell, β-adrenergic receptor agonist (β-ARA) stimulated tyrosine phosphorylation of IRS1 without significant change in tyrosine phosphorylation of insulin receptor β subunit. β-ARA enhanced insulin-induced tyrosine phosphorylation of IRS1 and serine phosphorylation of Akt. β-ARA phosphorylated Src, which may act as IRS1-tyrosine kinase. In conclusion, our data suggest that hypothalamic leptin-melanocortin-β-adrenergic system increases glucose uptake and insulin sensitivity in SM and BAT. Glucose uptake in SM appears to be associated with the enhancement of insulin signaling. Src kinase is a putative mediator for the enhancement of insulin signaling in response to β-adrenergic stimulation. [J Physiol Sci. 2008;58 Suppl:S104]

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Green tea contains high levels of polyphenols that are believed to prevent lifestyle-related diseases. The main tea polyphenols are tea catechins (TCs), which are thought to be useful compounds for the treatment of obesity. Although the mechanisms have not yet been determined, several studies suggest that TCs may reduce adiposity by inducing thermogenesis. To test this notion, we examined the effect of dietary TC intake on body fat accumulation in rats and uncoupling protein family (UCP1, 2 and 3) gene expression in brown adipose tissues (BATs). Male Sprague Dawley rats were fed for 5 weeks with a high-fat (HF; 35% fat) diet, then randomly divided into 4 groups and fed for 8 weeks with a HF, HF with 0.5% TC (HFTC), normal fat (NF; 5% fat), or NF with 0.5% TC (NFTC) diet. At the end of the experimental period, perirenal and epididymal white adipose tissues (WATs) and interscapular BAT were isolated. The NFTC group had significantly lower perirenal WAT weights than the NF group but the HF and HFTC groups did not differ significantly. TC intake had no effects on epididymal WAT weights. The NFTC group had significantly higher UCP1 mRNA levels in BAT than the NF group but the HF and HFTC groups did not differ significantly. TC intake had no effect on the UCP2 and 3 mRNA levels in BAT. These results suggest that the suppressive effect of TC on body fat accumulation in rat is closely associated with BAT UCP1 expression. [J Physiol Sci. 2008;58 Suppl:S105]

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Noradrenaline released from sympathetic nerve terminals causes lipolysis and thermogenesis via activation of β₃- adrenergic receptor, subsequent signaling and uncoupling protein (UCP) in brown adipocytes, regulating body temperature and energy balance. Watanabe et al. (2006, Nature, 439, 486) recently suggested cholic acid intake prevents obesity in high fat diet-fed mice via enhancement of the expression of D2, an enzyme that converts T₄ (thyroxin) to T₃. We have studied whether this preventive effect of cholic acid on obesity formation is caused by enhanced thermogenesis via β₃-adrenergic lipolysis and α₁- and β₃-adrenergic Ca²⁺ signaling. Changes in intracellular Ca²⁺ ([Ca²⁺]_i), mitochondrial membrane potential and heat production were recorded by fluorometry and thermography in mice fed with high fat diet, high fat diet with cholic acids or chow for 7-9 weeks. Expression of proteins and mRNA of adrenergic receptor were also examined. Abnormal increases in body weight by high fat diet were prevented by cholic acid intake. Cholic acids-fed mice showed a greater heat production, greater FCCP-, α₁- and β₃-adrenergic rises in [Ca²⁺]_i, tighter mitochondria-endoplasimic reticulum couplings and greater expression of α₁- and β₃-adrenoceptor proteins in brown adipocytes. Thus, cholic acid intake prevents high fat diet-induced obesity by increasing energy expenditure via increased adrenergic signaling in brown adipocytes. [J Physiol Sci. 2008;58 Suppl:S105]

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We investigated whether angiotensin II (ANG II) and its receptors contributes to lipopolysaccharide (LPS)-induced microglial activations through their effect on the activation of proinflammatory transcription factors, NF-kB and AP-1. To this end, examined were the effects of an ANG type 1 receptor (AT1) antagonist, losartan, on the LPS-induced productions of interleukin-1 (IL-1) and nitric oxide (NO), morphological changes of the cells, as well as the activations of NF-kB and AP-1 in primary culture of microglial cells. Our RT-PCR study revealed that LPS-stimulated microglial cells had marked expression of mRNAs for AT1 and angiotensinogen. LPS (100 ng/ml)-stimulated microglial cells showed increases in IL-1 and nitrite (a relatively stable metabolite of NO) concentrations, and in the expression of IL-1 mRNA, as well as a morphological change from an amoeboid shape to a multipolar (mostly bipolar, but sometimes tripolar) rod shape. These effects were all significantly inhibited by treatment with AT1 antagonist, losartan. The activity of NF-kB and AP-1 was enhanced in LPS-stimulated microglia, that were significantly suppressed by losartan. Application of ANG II itself enhanced the LPS-induced increase in nitrite concentration, that were inhibited by losartan. These results suggest that ANG II enhances microglial activities through the stimulation of microglial receptor, AT1, which evokes activation of transcription factors, NF-kB and AP-1. [J Physiol Sci. 2008;58 Suppl:S105]

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Syrian hamster (Mesocricetus auratus) hibernation is characterized by a lack of gene expression yet hibernators are responsive to various stimuli and arouse spontaneously from body temperature at near freezing to 37 °C (cenothermia) at regular intervals of about 100 hr. Brain metabolism including neurotransmitter production under extreme hypo-metabolic conditions during hibernation was investigated by using HPLC combined with microdialysis technique and ¹³C-NMR spectroscopy. Analysis of acid digested brain tissue demonstrated the production of ¹³C lactate within 1 hr of IV infusion of 1-¹³C glucose while GABA and glutamate were enriched by 24 hr. Temperature independent microdialysis demonstrated that extracellular levels of striatal amino acids in hibernation are 80 to 90% lower than levels at cenothermia. Application of ¹⁴C-labeled glucose into the striatum by microdialysis during hibernation confirms intracellular synthesis of lactate, glutamate and GABA. The diffusion of radioactive substances from the striatum during hibernation labels the ipsilateral hemisphere by 24 hr and the contralateral hemisphere by 60 hr. We detected reduced but still active biochemical pathways in spite of the small electrical activity of the neuronal tissue. Immediate production of lactate in response to glucose administration implies brain hypoxia which might be due to the left-shift of oxygen saturation curve of hemoglobin under low body temperature. The results imply that, during hibernation, neuron-astrocyte cycling of glutamine-glutamate is arrested, but intracellular synthesis of components of this cycle still functions. [J Physiol Sci. 2008;58 Suppl:S105]

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Food intake is controlled by signals from the brainstem that mediate signals from the gut. Prolactin-releasing peptide (PrRP) neurons are localized in the brainstem. In the present study, we investigated whether food intake activates PrRP neurons. We examined expression of phospho-CREB in PrRP neurons after food intake. The percentage of phospho-CREB-positive PrRP neurons in the nucleus of tractus solitarii was increased by food intake, suggesting that food intake activates PrRP neurons in the nucleus tractus solitarii. To further study the role of endogenous PrRP in food intake, we generated PrRP-deficient mice. PrRP-deficient mice developed late onset obestity associated with metabolic disorders due to hyperphagia but not to energy expenditure, and showed an attenuated response to the peripheral satiety signal, cholecystokinin. Blockade of endogenous PrRP signaling by a central injection of monoclonal anti-PrRP neutralizing antibodies increased food intake, reflecting an increase in meal size. Furthermore, we demonstrated that leptin-induced reduction in food intake and body weight was impaired in PrRP-deficient mice. All these data suggest that PrRP relays satiety signals within the brain and that disturbance of this system can result in obesity and associated metabolic disorders. [J Physiol Sci. 2008;58 Suppl:S106]

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Endocannabinoid is known to enhance food intake by binding to cannabinoid receptor 1 (CB1R). It was reported that expression of CB1R was suppressed by injection of cholecystokinin (CCK). The underlying mechanism, however, has not been clarified until the present time. To investigate whether inhibitory effect of CCK on food intake is mediated by CB1R, mRNA expression of CB1R, CB2R (cannabinoid receptor 2), leptin receptor short type (Ob-Ra) and long type (Ob-Rb) in hypothalamus were measured by RT-PCR method before and after 24 hr-fasting. These mRNA expressions were compared between CCKA receptor deficient male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and its normal counterpart of male Long-Evans Tokushima Otsuka (LETO) rats. The amount of daily food intake was measured before and after 24 hr-fasting. The expression of CB1R mRNA, but not CB2R mRNA, was significantly decreased after 24 hr-fasting in OLETF rats. The expression of CB2R mRNA in OLETF rats after 24 hr-fasting was significantly increased as compared with that in LETO rats. There were no significant differences of Ob-Ra and Ob-Rb mRNA expression between OLETF and LETO rats both before and after 24 hr-fasting. Amount of food intake at nighttime and daytime before fasting and at nighttime after fasting in OLETF rats were significantly increased as compared with those of LETO rats. These data suggest that CCK may influence the expression of CB1R mRNA in hypothalamus after 24 hr-fasting, thereby enhancing food intake. [J Physiol Sci. 2008;58 Suppl:S106]

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To investigate whether repeated forearm immersion into the artificial CO₂-rich water influences O₂ consumption (MO₂) and blood flow (BF_muscle) of forearm skeletal muscle, near-infrared spectroscopy (NIRS) was carried out. Eight healthy volunteers (3 female and 5 male, 21-22 years) seated in upright position were instrumented on the right forearm with a laser Doppler flowmetry probe for recording skin blood flow (BF_skin), a NIRS-probe for recording muscle hemoglobin (Hb) contents. Blood flow of the forearm was restricted by comprising the inflatable cuff on the upper arm to perform three consecutive venous occlusions (<50 mmHg for 20 s) followed by an arterial occlusion (<280 mmHg, 50 s). MO₂ was determined by evaluating the slope of regression line of oxy-Hb decrease during arterial occlusion, and BF_muscle was determined by the rate of increase of total-Hb during venous occlusion. The forearms of the subjects were immersed CO₂-water (860-990 ppm, right forearm) and tap water (CO₂<20 ppm, left forearm; control) at 30 °C for 15 minutes 5 times/a week for 2 weeks. The BF_skin doubled 2 weeks after repeated CO₂-water immersion, compared with tap water immersion. After 2 weeks repetition, MO₂ and BF_muscle of the arm immersed into CO₂-water was respectively 51% and 45% larger than those into tap-water. Results show that repeated immersion for 2 weeks into artificial CO₂-water may increase the muscle metabolisms in the local immersed. [J Physiol Sci. 2008;58 Suppl:S106]

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We investigated the modifications in monoamines [dopamine (DA) and serotonin (5-HT)] and their metabolites [3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA)] levels and caspase-3 activity in the brain of mice under bisphenol A (BPA) treatment. BPA was administrated throughout embryonic/fetal and infant stages through placenta and milk. At the age of 4 weeks, the male mice were sacrificed. BPA administration did not change body weight, but induced the decrease of wet weight of the brain. It induced the decreases of DA and 5=HT levels and the increases of (DOPAC + HVA)/DA and 5-HIAA/5-HT ratio in some parts of the mouse brain. The activity of monoamine oxidase B (MAO-B), an enzyme converts DA to DOPAC and HVA, was not changed by BPA administration. Caspase-3 is an enzyme activated during the cascade of events associated with apoptosis. No significant change of the caspase-3 activity was observed. There are some reports that BPA administration caused motor hyperactivity. This increase of monoamine turnover may result in hyperactivity. At the age of 4 weeks, apoptosis of nerve cells may not be induced by BPA administration. In this study, we demonstrated that BPA induces the underdevelopment of brain, hyperactivity of monoamine neurons and unchanged activities of MAO-B and caspase-3. [J Physiol Sci. 2008;58 Suppl:S106]

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Our previous study showed that estrogen modulated the activation of nitric oxide-producing neurons in the parvocellular paraventricular hypothalamic nucleus (pPVN) induced by cage-switch stress. In this study, we investigated whether estrogen affects the stress-induced activation of oxytocinergic and corticotoropin releasing hormone (CRH) containing neurons through nitric oxide in the pPVN in ovariectomized (OVX) rats by comparison of c-Fos expressions using immunohistochemistry. Nine-week-old female rats were ovariectomized and divided randomly into placebo-treated (OVX+Pla) and estrogen-treated (OVX+E2) groups. The rats were subcutaneously implanted with pellets containing 17 β -estradiol or a placebo 4 weeks after OVX. Four weeks later, the rats underwent cage-switch stress for 30 min with or without pretreatment of L-NAME. At 60 min after the cessation of this stress, the rats were anesthetized and perfused. The brain sections were processed for c-Fos and oxytocin, and CRH immunohistochemistry. Cage-switch stress increased activation in oxytocinergic neurons in the pPVN in OVX+Pla, but not in that of OVX+E2, rats. These findings suggest that estrogen may attenuate the activation of oxytocinergic neurons, via at least in part, nitric oxide. [J Physiol Sci. 2008;58 Suppl:S107]

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We examined whether acute psychological stress enhances plasma oxidative stress, which is restored by estrogen replacement in ovariectomized rats using nitrotyrosine (NT) as a stress marker. Female Wistar rats aged 9 wk were ovariectomized and treated with placebo or 17β-estradiol (1.5 mg/60-day release, sc) pellets 4 wk after ovariectomy. Rats aged 16 wk were catheterized for blood sampling. Six days post-surgery, rats were exposed to cage-switch or restraint stress and blood samples were collected during the stress and the recovery from the stress. Basal plasma NT level was significantly higher in the placebo-treated rats than in the estrogen-treated rats, though the basal plasma nitric oxide metabolites (NOx) level was similar in both groups. The cage-switch stress significantly increased the plasma NT concentrations, and decreased the NOx levels during the stress and the recovery only in the placebo-treated rats. The stress had no effects on the plasma NT and NOx levels in the estrogen-treated rats. These results suggest that estrogen replacement attenuates the excess oxidative stress and restores NO bioavailability during the psychological stress and the recovery. [J Physiol Sci. 2008;58 Suppl:S107]

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There are many patients who are suffering from chronic muscle pain, however, neural mechanisms for it were not well understood. Previously we reported that animals exposed to RCS (4ºC) showed muscle mechanical hyperalgesia. However, effect of RCS to thermal nociception has not been studied yet. Furthermore, different temperature (4ºC or -3ºC) was used in different reports, but the difference in effect of used temperature is not clear. Therefore we examined effects of RCS on thermal nociception and effects of different exposure temperature. Rats were exposed to RCS (from room temperature to 4ºC or -3ºC for 30 min every an hour from 10 am to 5 pm, and 4ºC or -3ºC from 5 pm to 10 am on the next day) for 5 days. Deep mechanical hyperalgesia of the lower leg in both sides was observed up to 42 days after RCS at -3 ºC, and cutaneous mechanical hyperalgesia was also observed. The thermal hyperalgesia was observed 1, 4 , 21, and 28 days after RCS. In contrast, RCS at 4 ºC induced deep mechanical hyperalgesia up to only 21 days, thermal hyperalgesia for a shorter period, and no cutaneous mechanical hyperalgesia. These results suggest more severe RCS (-3ºC) can induce longer lasting mechanical hyperalgesia in the muscle, and additionally it can induce cutaneous mechanical and thermal hyperalgesia. Hyperalgesia by RCS has been considered to be induced by impaired descending pain inhibitory system. Taking this into consideration, present observation suggests that descending pain inhibitory system more strongly inhibits deep nociception than skin nociception. [J Physiol Sci. 2008;58 Suppl:S107]

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We previously reported that levels of plasma corticosterone of rats in the alternatively changing ambient temperature (repeated temperature changes between 4 degrees and 27 degrees, each lasting for 1h) were higher than those in the constant low ambient temperature (4 degrees) at 1, 2, 4 and 10 days after the exposure. Frequently changing, but not constant, ambient temperatures may have induced a stronger stress. Furthermore LPS-induced fever in a group with high level of plasma corticosterone was enhanced. Plasma levels of TNF-α 3 h after LPS ( i.p. ) in animals exposed to constantly low and alternatively changing ambient temperatures for two days, tended to be higher than those exposed to constant 25 degrees temperature. Plasma levels of endotoxin in animals exposed to alternating ambient temperatures were higher than those exposed to constant 25 degrees and constant 4 degrees at 12 h and 2 days after the exposure. The possibility was considered that intrinsic endotoxin from enterobacterum leaked by stressful ambient temperature, and that leaked endotoxin primed macrophage resulting in enhanced fever. The aim of the present experiments was to ravel underlying mechanisms of the enhanced LPS-induced fever in animals exposed to stressful ambient temperatures. For this purpose, TNF-α production from rat peritoneal macrophages after the each ambient temperature exposure was observed, and the possibility of priming the cytokine productivity of macrophage by the intrinsic endotoxin is examined. [J Physiol Sci. 2008;58 Suppl:S107]

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Purpose: The university student chiefly takes an examination of the national exam for clinical laboratory technician at the end of the university. It is difficult to look for the student who suffers from the academic (=mental) stress because there is a few index with a good academic stress. Then, this examined it by using some stress indices aiming to know the method that was able to be passed by a lot of students in this research.Methods: Subjects: 28 healthy student volunteers were given informed written consent. Chemical stress index: Levels of Diacron-Reactive Oxygen Metabolite: d-ROMs (FRAS-Free Radical Analytical System;Italy). Psychological evaluation: Affect grid developed by Russell JR, Weiss A and Mendelsohn GA. Measurement time: Before a week National examining.Results: Blood d-ROMs concentrations were not increased significantly normally from 250±49U.CARR a week before. In Affect grid, both the score of "Pleasantness" and "Awake" were normal ranges. However, there was correlation between the result of the examination (order) and the blood d-ROMs concentration (r =0.497, p<0.01).Conclusions: The result of the person with blood d-ROMs concentration examination before a week tended to be normal. It was suggested that the measurement of the blood d-ROMs concentration about one week before the examination for making a lot of students pass the examination and caring mentally be effective. [J Physiol Sci. 2008;58 Suppl:S108]

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Exposure of humans to microgravity condition resulted in body fluid shift and myatrophy. The aim of this study was to test the effectiveness of artificial gravity and ergometric exercise as countermeasure to these changes. 12 healthy young men were exposed to stimulated microgravity for 20 days by head-down bed rest. 6 subjects randomly selected were subjected to 1.4G of artificial gravity with 60W of ergometric workload everyday for 30 minutes (CM group). The rest of the subjects served as the control (control group). In head-up tilt test and Anti-G test, body fluid shift measured by segmental bioimpedance method was smaller in the trunk in CM group than in control group. Also, myatrophy measured by MRI in the thigh was smaller in CM group than in control group. Artificial gravity with exercise appeared to be effective in preventing changes in body fluid shift and myatrophy due to microgravity exposure. [J Physiol Sci. 2008;58 Suppl:S108]

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Effectiveness of centrifuge-induced artificial gravity (AG) and ergometric exercise (EE) as a countermeasure to space deconditioning, including cardiovascular deconditioing, myatrophy, and bone-mineral metabolism, induced by 20 days of head-down bedrest,(HDR) was examined in 12 healthy men. HDR was performed with 2300 kcal of diet. Water intake was recommended more than the urine output in the previous day. A new protocol of gravity-exercise step-up protocol up to 30 min/day was adopted, with 1.6 G of AG and 60W of EE at the initial load every day. AG was increased by 0.2 G or EE load was increased by 15W when the subject approved the step-up. The effectiveness of this protocol will be discussed. [J Physiol Sci. 2008;58 Suppl:S108]

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We reported previously that a transient drop in arterial blood pressure (ABP) was induced by head down rotation (HDR) in urethane-anesthetized rabbits. Our results suggested that vestibular inputs due to HDR played a role in the drop of ABP possibly through sympathetic nerve outflows. However, the anesthetic agent may have influences on control of ABP and vestibular inputs. Accordingly, it is important to examine ABP changes during HDR using conscious animals. A week before the experiment, a catheter was inserted from the left carotid artery and was positioned in the aorta at the level of the aortic arch to monitor blood pressure. Chemical labyrinthectomy was applied by injecting 20 mg/kg sodium arsanilate into the bilateral intratympani two days before the experiment. In control rabbits, the same amount of saline was injected instead of the sodium arsanilate. On the experiment day, the animal was mounted comfortably on a tilting table in a dark room. The animal was tilted to 45-degree head-down within 5 s and kept at the position for 1 minute. HDR increased the ABP (from 108 ± 4 mmHg to 118 ± 8 mmHg) and kept the pressure at the increased level until the end of HDR in the control alert rabbits. HDR in vestibular-lesioned (VL) rabbits increased the ABP from 103 ± 6 mmHg to 120 ± 7 mmHg. The increase in VL rabbits (117 ± 3%) was significantly larger than that (110 ± 3%) in controls. These results suggest that vestibular organs are involved in the control of ABP during HDR. [J Physiol Sci. 2008;58 Suppl:S108]

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During pregnancy, physical and mental conditions are greatly changed. Changes in body alignment will affect the standing posture. Also, anxiety generally increases as the week of gestation precedes. We have recently evaluated the differences in postural control during stance between pregnant and non-pregnant women (Nagai et al., 2008). In the present study, we examined the influence of anxiety on postural control in pregnant women. Body sway during upright standing and state-anxiety (STAI-S) on the day of experiment were measured in pregnant (n = 35) and non-pregnant (n = 33) population. Each population was further divided into two groups on the basis of the median split of the distribution of STAI-S scores. The data was analysed among 4 groups, pregnant with high anxiety, pregnant with low anxiety, non-pregnant with high anxiety, and non-pregnant with low anxiety. A positive correlation (r = 0.54) was found between state-anxiety and enveloped area of body sway in pregnant group with high anxiety. Two way ANOVA (pregnancy x anxiety) showed that the enveloped area in pregnant population was greater (p < 0.02) than that in non-pregnant population. Our findings suggest that pregnancy increases body sway during upright standing and the influence of pregnancy is apparent in high anxiety population. [J Physiol Sci. 2008;58 Suppl:S109]

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In pregnant women, standing posture changes as the fetus develops. Increasing load onto the lumbar spine and abdominal muscles causes a shift of the head position in the posterior direction and increase the lumbar lordosis and anterior pelvic tilt. In the present experiments, we have tried to identify the characteristics of postural control during orthostatic standing in pregnant women in comparison to age-matched non-pregnant women by analyzing posturograms. The week of gestation in pregnant women was 30.3 on average, and body weight and body mass index (BMI) were greater in pregnant women than those in non-pregnant women. In pregnant women, the area of body sway during upright standing for 1 min was greater, by 88% with eyes open and by 43% with eyes closed, than that in non-pregnant women. A fast Fourie's transform analysis of body sway showed that the spectral power of frequency band over 1 Hz was smaller in pregnant women, by 31% with eyes open and by 34% with eyes closed, than in non-pregnant women. Body sway of over 1 Hz is generally stabilized by somatosensory inputs, therefore, the result indicates that influences of physical changes, represented by body weight and BMI, are minimized in pregnant women by improving the sensitivity and/or accuracy of information processing of somatosensory inputs. [J Physiol Sci. 2008;58 Suppl:S109]

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This topic was designed in a series of studies to speculate on the human reproduction possibly performed in the space environment in future. The reproduction is one of important factors of sexuality which is a fundamental property to construct a new human society in space. To clarify the effect of gravity on the development of fetus in the uterus we tried to analyze the relation of movements of the pregnant woman to the posture of her fetus using a three dimension ultrasonic diagnostic device (VOLUSON 730 Expert) and a two dimension dynamic image analysis software (Move-tr 2D) for computing. Healthy pregnant women and their fetuses were passively tilted from the standing position to the supine position or vice versa on a tilt table. An echo probe was placed on the lower abdominal wall of the mother, visualizing movements of the uterus and fetus. The relation of both movements was expressed with changes in a relative angle between two lines which were imaged on each of the uterine wall and fetus, and a trendgraph of changes in posture of the fetus was obtained. From the results it seems that the fetus moves independently to take its original posture against gravity, although it moves in accordance with mother's movement immediately after the start of tilting [J Physiol Sci. 2008;58 Suppl:S109]

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Respiration induced heart rate variability (RHRV) was only found in mammals and avian showing a remarkable postnatal development, whereas no RHRV in aquatic species such as fish or amphibian. To elucidate our hypothesis that gravity exposure may plays a key role in the postnatal development of RHRV as well as its evolutional origin, we have studied effects of hypergravity (2G) on the postnatal development of RHRV using rat. Pregnant Wister rats were kept in centrifugal cages for 38days from 6 gestational days to 23 postnatal days. Electrocardiograms were recorded from the neonates in 2 to 23 postnatal days in 2G group with simultaneous control (1G) group. The RHRV analysis was performed by calculating a component of Fourier power spectra coincide with the respiration frequency range. In both groups, averaged resting heart rate gradually increased during the period. When comparing the heart rate between the two groups, 2G group indicated significantly lower than 1G control (p<0.001) at 2 days old, where as no significance at 23 days old. The RHRV of 2 days old neonates in both groups indicated very small magnitude, but significantly lower in 2G group than 1G control (p<0.01). The RHRV was not increased during the first 2 weeks and then sharply increased to reached 45 fold in magnitude at 23 days for 1G control, whereas 69 fold for 2G group. The results strongly suggested that the postnatal innervation from respiration to cardiovascular centers was gravity dependent. [J Physiol Sci. 2008;58 Suppl:S109]

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On Earth, signals from otolith organs can be interpreted either as linear motion or as tilt with respect to gravity. In microgravity, static tilt will no longer give rise to changes in otolith activity. However, linear acceleration as well as angular acceleration stimulates the otolith organ. Therefore, during adaptation to microgravity, otolith-mediated response such as eye movements would alter. Flatfish provide a natural model for the study of adaptive changes in the vestibulo-ocular reflex. During metamorphosis, vestibular and oculomotor coordinate of flatfish displaced 90 degrees about the longitudinal body axis. Therefore, it is expected that microgravity induce the sensory mismatch in adult flatfish. In this study, we analyzed the eye movements for body tilting in normal flatfish and in unilaterally otolith removed flatfish. The eye movements for body tilting along the different body axis were video-recorded. The vertical and torsional eye rotations were calculated from the images digitized by computer.In normal flatfish, the properties of vertical and torsional eye movements for body tilt were almost the same as that in goldfish. After removal of left utricular otolith, the amplitude of vertical eye movements decreased. Especially for 180 degrees tilting, the vertical eye movements almost disappeared. These results suggested that utricular otolith play some roll in flatfish. [J Physiol Sci. 2008;58 Suppl:S110]

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We examine that the contribution rate of cardiac output and muscle sympathetic nerve activity (MSNA) on orthstatic torerance induced by simulated weightlessness using 20 days head-down bed rest. The subjects lay on the tilt table at the 0° horizontal position and was then inclined to the 15° head-up, 30° head-up and 60° head-up position. The tilt table remained in each position for 15 min. MSNA was recorded from the tibial nerve at the popliteal fossa with tungsten microelectrode. [J Physiol Sci. 2008;58 Suppl:S110]

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Cardiovascular function is most vulnerable when exposed to high positive G acceleration in the z-axis (+Gz). The purpose of the sturdy is to evaluate cardiac micro-injuries on +Gz stresses. Anesthetized male SD rats were divided into three groups: control (CONT), +Gz exposure with an anti-G suit (WS), and +Gz exposure without an anti-G suit (WOS). Rats of the WS and WOS groups were exposed to +5Gz stress twice with 30-minute interval by the centrifuge. Arterial pressure level of the brainstem (APLB), heart rate and central venous pressure were recorded. Blood samples were taken three hours later after the second +Gz exposure. Plasma concentration of cardiac troponin T (cTnT) was measured by the electrochemiluminescence immunoassay technique. During +Gz exposure, severe hypotension was found in the WOS rats, while APLB was sustained in the WS rats. Plasma cTnT in the CONT, WS or WOS group was 0.086 ± 0.034, 0.502 ± 0.034 or 0.498 ± 0.076 ng/ml (mean ± SE), respectively. Plasma cTnT was significantly higher in both of the +Gz exposure groups than in the CONT group. No significant difference was found in plasma cTnT between the WS and WOS groups. +Gz stress caused severe hypotension in a brain level in the WOS rats, but not in the WS rats. The plasma cTnT concentration was increased by +Gz exposure in both of the WS and WOS groups. The result suggests that the +Gz stress per se may cause cardiac injury even when an anti-G suit functions well. [J Physiol Sci. 2008;58 Suppl:S110]

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High sustained +Gz stress has been known to provoke G induced loss of consciousness and central nervous system insults. Direct recording of tissue PO₂ in the brain during +Gz exposure has not yet been reported. The purpose of this study is to evaluate effects of +Gz stress in the brain PO₂. Anesthetized male SD rats were exposed to +3Gz by a centrifuge with an anti-G suit. Arterial pressure level of the brainstem (APLB), central venous pressure, heart rate and PO₂ of the brain were measured. Cortical and hippocampal PO₂ were simultaneously measured by a PO₂ monitor with the polarographic oxygen electrodes. A decrease in APLB was observed twice: during the phase of increasing +Gz and the phase of decreasing +Gz. At these lowest points, cortical and hippocampal PO₂ were also decreased significantly (first: 83.0 ± 2.4% and 68.8±4.4%; second: 81.0 ± 1.6% and 71.7 ± 6.7%; vs. control). APLB was restored to the control level just after the +Gz exposure was stopped. However, cortical PO₂ was not restored to the control level over 5 minutes after the +Gz stop, although hippocampal PO₂ was restored until 1 minute. +Gz induced hypotension involved a decrease in brain-tissue oxygen concentration. However, the time course of the PO₂ changes during the +Gz exposure were different between cortex and hippocampus. This result may describe that the +Gz effect on the brain oxygen concentration is different dependently on the brain areas. [J Physiol Sci. 2008;58 Suppl:S110]

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In the clinical setting, blood pressure measurement with mercury sphygmomanometer may often be disturbed during tucking up of the sleeves. Tendency of such errors in various studies, however, is not consistent. In the present study, we attempt to clarify the mechanism of disturbed blood pressure measurement using controlled-constriction method. Bilateral simultaneous blood pressure measurement on bilateral upper arms by two experts using sphygmomanometers were carried out after confirmation of similarity of bilateral blood pressures in healthy female subjects (n=16, 21±1 yr). In the measurement, one of the upper arms was constricted with a band by 1cm or 2cm shorter than circumference of upper arm at the axillary level. There were no statistical significance between control values and the values obtained from the arm of 1cm-constriction. However, during 2cm constriction, systolic pressure at constricted arm was measured 105±7 mmHg that was significantly lower than the control value (108±7 mmHg) and diastolic pressure at constricted arm was 69±8 mmHg that was higher (p<0.05) than the control (66±7 mmHg). These results suggest that constriction of upper arm may increase the space of Windkessel in the pre-constriction region of the arteries where more blood of stroke volume is stored during systole, which leads to the lower systolic pressure measurement. Subsequently, the more blood is flowing from the Windkessel during diastole, which leads to the higher diastolic pressure measurement. [J Physiol Sci. 2008;58 Suppl:S111]

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ATP is released from various cells by specific stimulus and cellular damage, and serves as a chemical mediator/transmitter for intercellular signaling. Upon a mechanical stimulus, cultured epidermal keratinocytes release ATP, which is proposed to transmit the mechanical information to sensory nerve terminals in the skin. Because heat-sensitive ion channels, TRPV3 and V4, are expressed in keratinocytes, we hypothesized that heat also causes ATP release from keratinocytes to transmit the thermal signal to neurons or elicit other biological events in the skin. To assess whether heat causes ATP release from the skin, excised mouse scrotal skin was superfused with Krebs-Henseleit solution in a small bath, and ATP concentration in the superfusate was measured by luciferin-luciferase assay. Upon heat application up to ∼42°C, two types of heat-evoked ATP release were observed: one is slowly activated from lower temperature (∼29°C), and another is steeply activated at higher temperature (∼40°C). The latter response only was significantly reduced by ruthenium red (RR, 50 μM), a wide-range inhibitor for TRPs. The RR-sensitive ATP release was activated at near the activation temperature of cultured keratinocytes (Tominaga et al., J. Physiol. Sci., 57, Suppl., S99, 2007). These results support that heat causes ATP release from the skin by the mechanisms involving TRPV3/V4 or other TRPs. ATP might have a role in intercellular signaling of temperature-sensitive events occurring in the skin. [J Physiol Sci. 2008;58 Suppl:S111]

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We examined whether skin sympathetic nerve activity (SSNA) was involved in reduced cutaneous vasodilation in hypovolemic hyperthermia. Seventeen young men were divided into normovolemia (N, n=8) and 9% hypovolemia with diuretics (L; n=9). After 5-min baseline measurement, they were warmed for 45min with a perfused suit until esophageal temperature rose by 0.4°C, while SSNA (microneurography; the peroneal nerve), skin blood flow (SkBF; laser-Doppler flowmetry; the dorsal foot), and mean arterial pressure (MAP) were recorded. Although a rising rate of cutaneous vascular conductance (CVC=SkBF/MAP) during warming was 50% lower in L than N (P<0.05), increasing rates of burst frequency and amplitude of SSNA, rectified and filtered with low pass edge of 1.6Hz, were similar between N and L. Next, to assess the relationship between SSNA firing rhythm and cardiac cycle, an incidence of original SSNA spikes was counted every 0.1s bin for 3s after every R wave of ECG. In an averaged histogram of spike incidence every 1min, the peaks appeared at the same interval as those of R and the peak interval of spike (I_S) was highly correlated with that of R (I_R) in N (n=399, r=0.92) and L (n=443, r=0.78) for 50 min (both, P<0.001). The standard deviation of [I_R-I_S] (SD_R-S)in L was 36% higher than N (P<0.05). Moreover, SD_R-S was inversely correlated with integrated change in CVC (r=-0.64, P<0.01). Thus, increased SD_R-S may be linked with reduced cutaneous vasodilation in hypovolemic hypovolemia. [J Physiol Sci. 2008;58 Suppl:S111]

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We have previously demonstrated that rhythmic exercises such as Zen abdominal breathing, gum-chewing, hula dancing and so on caused an increased serotonin(5-HT) in adult human. In present study, we evaluated change in urinary 5-HT in 3-6y.o children before and after rhythmic exercise program (REP) including zigzag run, jumping rope, vaulting box, climbing bar, doing horizontal bar and overhead ladder etc. REP elicited a significant increase in urinary 5-HT in senior (5-6y.o.) children and smaller increase in junior (4-5y.o.) children. However mean basal level of urinary 5HT obtained before REP in senior children was lower than that of junior children. We further evaluated those modulating factors of season and fatigue which would affect REP-induced changes in urinary 5-HT. Fatigue, elicited by a whole day excursion, caused an attenuation of the REP-induced increase in urinary 5-HT. A seasonal effects were examined on a day in summer(27°C), winter(9°C) or spring(12°C). The mean basal urinary 5-HT obtained on the summer day was significantly higher than those on the winter day or spring day. REP on sunny day in winter caused greater increase in urinary 5-HT than that on cloudy day in winter. [J Physiol Sci. 2008;58 Suppl:S111]

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Background: We and others have reported from morphological examination that smooth muscle-like cells may be differentiated from bone marrow stromal cells (BMSCs). However, few studies have addressed whether the differentiated smooth muscle-like cells (BMSC-SMCs) also possess the functional properties of SMCs. We conducted physiological study to characterize BMSC-SMCs. Methods and Results: We assessed voltage-gated Ca²⁺ currents using whole-cell patch clamp methods. Whereas only 10% of BMSC-derived SM progenitor cells showed T-type Ca²⁺ channel current, 40% of BMSC-SMCs showed T-type Ca²⁺ channel current and other 60% showed L-type Ca²⁺ channel current. We also measured agonist-evoked [Ca²⁺]_i transients using Fura-2 imaging. Ca²⁺ transients was observed in BMSC-SMCs in response to SMC-specific agonists such as bradykinin (10⁻⁶ M) and angiotensin II (10⁻⁷ M). Finally, we assessed agonist-evoked contraction using video camera. In response to SMC-specific agonists, BMSC-SMCs showed contraction-like movement, i.e., change in shape and sequential movement. Conclusions: BMSC-SMCs exhibit functional and physiological properties of SMCs. Since BMSCs have the potential to differentiate into functional SMCs, they can be reliable and expandable SMC sources for the construction of tissue-engineered vascular grafts. [J Physiol Sci. 2008;58 Suppl:S112]

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The aim of this study was to examine on the mechanisms of the water secretion in the submandibular glands acini of the normal and xerostomia. Male Wistar rats, weighing 250-280 g, were used. Experimental animals were divided into two groups, normal and xerostomia rats. Glycopyrrolate was administered to induce acute xerostomia, we raised both anti-AQP5 antibody and the anti-junctional proteins (ZO-1) antibody, and examined the localization of these proteins in rat submandibular gland acini. And permeability test of the tight junctions of acini were examined cytochemical, using HRP as a tracer. Salivary flow rate evoked by pirocarpine stimulation was decreased in xerostomia rats to about 50% of the normal rate. In the normal gland, the immunostaining for AQP5 was intense along the luminal membrane and was less intense on the basal membrane, and it was not detected on the lateral membrane in the acinar cells. ZO-1 was detected on the basolateral membrane. In the xerostomia rats, localization of AQP5 was decreased comparing with normal rats. And ZO-1 was disappeared in the essential portion. In the normal gland, tight junctions were impermeable to HRP indicating an adequate tight junction seal, but the tight junctions in the gland of xerostomia rats were permeable to it. Because the water-rich fluid is secreted in the lumen of the acinar cells where both AQP5 and tight junctions were abundantly present, it is reasonable to speculate that both intracellular and paracellular transport play an important role in the water secretion concomitantly. [J Physiol Sci. 2008;58 Suppl:S112]

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Fish CNS neurons can repair their axons following nerve injury, while mammalian CNS neurons lose this capacity. After fish optic nerve transection, the regenerating optic axons can reinnervate the tectum by 3-5 weeks. We have investigated these regeneration mechanisms using goldfish and zebrafish visual system. Galectin is a family of carbohydrate-binding protein with an affinity for β-galactoside. It's noted that galectins mediate various biological functions such as cell-cell interactions, inflammation and cell adhesion by cross-linking of carbohydrate residues. In the present study, we examined expression of two members of galectins, galectin-1 and galectin-3 in adult fish retina during optic nerve regeneration. The level of galectins protein rapidly decreased within a few days, but thereafter increased by 10-20 days and returned to the control level by 30 days after optic nerve lesion. Analysis by RT-PCR and in situ hybridization revealed that level of galectin mRNAs started to increase within a few days, peaked at day 5-10, and then gradually decreased by day 30 after optic nerve transection. To elucidate molecular involvement of galectins, we estimated the effect of recombinant galectin protein on neurite outgrowth in retinal explant culture system. Recombinant galectin protein clearly induced neurite outgrowth from adult fish retinal explants. These data suggest that upregulation of galectins (galectin-1 and galectin-3) is an important event for regeneration of the optic axons. [J Physiol Sci. 2008;58 Suppl:S112]

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The fish is one of the most useful animal model for central nervous system (CNS) development and regeneration studies. Morphological and molecular biological knowledge has been piled up day by day. However, functional study, especially behavioral knowledge has not been fully studied. Therefore, accurate and simple quantification of fish behavior has become more required. The optomotor response has been used in the studies of visual development. Here, we developed a computer image processing system to analyze optomotor response in zebrafish during development and optic nerve regeneration. The opotomotor response of adult fish with optic nerve transection was recovered by 25-30 days after axotomy. On the other hand, the optomotor response in newborn zebrafish showed an adult pattern by 20 days after fertilization. Furthermore we applied this system to analyze behavior in transgenic fish during development. In the gene knock down embryos which were injected with anti-sense oligonucleotide of purpurin, a retina-specific regenerating molecule, the optomotor response was transiently inhibited in an early period of retinal development. Thus, this system is very powerful tool for studying behavior of development and regeneration in fish. [J Physiol Sci. 2008;58 Suppl:S112]

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Recently, we reported that purpurin is a retina-specific secretory protein and acts as a trigger molecule for axonal outgrowth during fish optic nerve regeneration. In zebrafish retinal development, although there was the highly expression of purpurin, the function of purpurin is not so clear. Here, we investigated the role of purpurin revealed by loss of function of purpurin gene using morpholino antisence oligonucleotides. During retinal development, cell cycle and neurogenesis is stringently controlled by several cues, and six types of neurons and one type of Muller glial cells were organized into distinct layers at 3 days post fertilization (dpf). However retinal lamination was not detected in purpurin morphant at 3 dpf. Muller cells were confined only inner retina and differentiation of photoreceptor and bipolar cells were not observed. Additionally, almost all retinal progenitor cells continued dividing even at 3 dpf. However, the inhibitory effect of morpholino was strictly transient, and purpurin mRNA and protein were normally detected at 5-7 dpf. At 7 dpf, we observed differentiated photoreceptor cells and bipolar cells. The visual function revealed by optomotor response was significantly retarded in purpurin morphant at 5 dpf. At 10 dpf, optomotor response was not changed between control and purpurin morphant. These results strongly indicate that purpurin is a key molecule for retinal neurogenesis in developing zebrafish. [J Physiol Sci. 2008;58 Suppl:S113]

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Zfp238 is a C(2)H(2) type zinc finger protein, containing POZ domain at N-terminal and zinc finger domain (ZFD) at C-terminal. RP58 mRNA expression is detected at embryonic day (E) 10 in the neuroepithelium, and subsequently in the ventricular zones of the cerebral cortex in the E12 embryo. Strong expression is observed in the preplate in the cerebral cortex from this stage onward. High levels of expression continued to be detected in the cortical plate and subventricular zone of the neocortex, hippocampus, and parts of the amygdala, but not in the thalamus or striatum. The expression is also observed in the adult mouse neocortex, hippocampus, parts of the amygdala, and granule cells in the cerebellum. Double in situ hybridization using GAD67 or VGLUT1 probes reveal that Zfp238 is expressed in glutamatergic excitatory neurons. To characterize the Zfp238 protein, we made rabbit polyclonal antibody against the sequence of RP58 without POZ and ZFD, which could recognize this protein specifically, but not isotype and truncated type. Zfp238 was ubiquitously expressed in various tissues including nervous system: pancreas, heart, testis and lung etc. Western blotting analysis using the embryonic and adult brain lysate showed that the detected bands were higher than the predicted size of this protein. The results suggest that Zfp238 is modified in some ways, and we are analyzing its mechanism and biological roles. [J Physiol Sci. 2008;58 Suppl:S113]

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The opening of mouse vaginal cavity to the skin is a postnatal tissue remodeling, occurring around the fifth week of life, which depends on a hormonally triggered apoptosis at the vaginal mucosa in the lower part of vagina. However, the exact mechanism of apoptosis remains unclear. Our recent analyses disclosed that mice lacking the class IV semaphorin, Sema4D develop vaginal atresia. TUNEL assay performed to examine the mechanism revealed significantly less apoptotic cells in vaginal mucosa of Sema4D-deficient mice compared to wild-type mice. A rescue experiment placing back recombinant Sema4D to primary vaginal epithelial cells from Sema4D-deficient mice clearly demonstrated that Sema4D induced a significant apoptosis of vaginal epithelial cells in caspase-3 dependent manner. Furthermore, SiRNA-mediated knockdown experiment revealed a crucial involvement of B-type plexin in the apoptosis-inducing activity of Sema4D. Thus these results indicate a non-redundant role of Sema4D in the tissue remodeling occurring around 5-week-old mice. As a comparative study, we also present immunohistochemical studies disclosing specific expression patterns of Sema4D and plexins in the human female genital organs. [J Physiol Sci. 2008;58 Suppl:S113]

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Age-related impairment (AMI) is one of the functional senescence of brain and does not accompany pathological symptom. Previously, we have identified that DC0 mutation is a suppressor of AMI in Drosophila. DC0 gene encodes a PKA (cAMP-dependent kinase) catalytic subunit preferentially expressed in the mushroom bodies (MBs), essential structures for olfactory learning and memory. Heterozygous mutations of DC0 gene, DC0/+, delay AMI more than two-fold without affecting lifespan. PKA activity in DC0/+ mutant is reduced to ∼60% of that in wild-type flies. Furthermore inhibition of PKA activity in the MBs of wild-type flies also suppresses AMI, indicating that activity of cAMP/PKA leads to AMI. Prevailing model of aging propose that functional senescence is caused by an accumulating processes such as by reactive oxygen species and insulin signaling. Using temporally and spatially restricted induction system of transgene, we here examined whether accumulative activity of cAMP/PKA signaling during aging is a causal factor of AMI. Surprisingly, in addition to constitutive expression of DC0 transgene during aging, AMI was restored in DC0/+ flies when DC0 transgene was acutely induced at old age. Furthermore, when induction of DC0 transgene was ceased in DC0/+ flies at old age, their memory was improved to young control level. These results demonstrate that activity of cAMP/PKA at old age, but not cumulative activity, gives rise to AMI. [J Physiol Sci. 2008;58 Suppl:S113]

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The subfornical organ (SFO) is related to drinking behavior and cardiovascular response, and many SFO neurons are excited by nicotine. Nevertheless, it has been reported that central nicotinic stimulation does not seem to induce drinking behavior but cardiovascular response. To explore the discrepancy, water intake following intracerebroventricular (ICV) injection of nicotine and sensitivities for nicotine in SFO neurons were investigated in rats in detail, compared with effects of angiotensin II (ANG), which is well known as a dipsogenic material. Latency to drinking by ICV injection of nicotine at 1-100 nmol was dose-dependently shortened, but still total amount of water intakes was lower than that by ICV injections of ANG at 1-100 pmol. In extracellular recording from SFO slice preparations, 57% of SFO neurons were excited by both NIC at 30 μM and ANG at 100 nM. The neural excitation by nicotine was obviously transient while that by ANG was long-lasting. Our electrophysiological classification of dissociated SFO neurons in whole-cell patch-clamp recording showed that most nicotine-sensitive neurons were classified as F-type, a half of which showed ANG-sensitivity. These results suggest that nicotinic activation of SFO neurons is involved in a neural network of the brain triggering drinking behavior. [J Physiol Sci. 2008;58 Suppl:S114]

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Oxidative stress is enhanced in regular hemodialysis (HD) patients. Oxidized albumin is a reliable marker of oxidative stress. Depending on redox state, there are three major fractions of human serum albumin (HSA); HMA (reduced form) with a free thiol group on cysteine-34, HNA-1 (reversible oxidation product) with cystine, HNA-2 (irreversible oxidation product) with more oxidized state. We have studied the albumin thiol oxidation and the serum protein carbonyl formation in 21 HD patients. Albumin fractions were analyzed by HPLC and carbonyl content of protein (CC) was determined by Protein Carbonyl Assay Kit. The mean values for f (HMA) pre- and post-HD session were 46.6 ± 8.5% and 66.0 ± 8.9% respectively. In contrast, f (HNA-1) was significantly decreased the course of the HD session (48.2 ± 8.2% at pre-, 30.3 ± 8.3% at post-HD), and f (HNA-2) was also decreased (5.2 ± 1.0% at pre-, 3.7 ± 0.8% at post-HD). CC was also decreased (0.9 ± 0.2 nM/mg protein at pre-, 0.7 ± 0.2 nM/mg protein at post-HD). In relationship between the thiol oxidation and the carbonyl formation, value for CC was not correlated with that for HNA-1 (R = 0.3, P = 0.06) but significantly with that for HNA-2 (R = 0.6, P < 0.0001). We found the close relationship between irreversible oxidation of HSA and serum protein carbonyl formation in HD patients. [J Physiol Sci. 2008;58 Suppl:S114]

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A descriptive cross sectional study was conducted to assess the causes, socio-demographic factors and the management of anemia in patients with CKD, admitted to the Dialysis Units of NHSL, Sri Jayawardenepura and Western Infirmary Hospitals. Ethical clearance was obtained from the Ethical Review Committee of the Faculty of Medicine, Colombo and NHSL to collect data from medical records of patients (N=150).Most of the patients were between 35 to 55 years and 72.6% were males. Our study represented most of the districts in Sri Lanka, the majority (55.3%) being from Colombo. Hypertension (27.3%) was identified as the leading cause in both males and females, while diabetes (21.3%) was the second commonest. 10.7% patients had both hypertension and diabetes. Other causes comprised of glomerular nephritis, polycystic kidney disease, obstructive kidney disease, SLE and Alport^'s syndrome. In 20% of patients the cause was not yet identified. 10% were found to have severe anemia and the main mode of management was blood transfusion. Later they were given recombinant erythropoietin (rEPO). 60% had moderate anemia of which 65.6% had received rEPO while 17.8% had not received any treatment. The commonest causes leading to Chronic Kidney Disease are hypertension and diabetes. Main treatment option of anemia was rEPO which was started mostly at hemoglobin levels less than 9g/dl, but according to DOQI guidelines it should be commenced when hemoglobin level is 11g/dl. [J Physiol Sci. 2008;58 Suppl:S114]

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Cyclooxygenase-2 (COX-2) is the key enzyme regulating the production of prostaglandins (PGs), and is expressed in the kidney macula densa (MD) cells. COX-2-derived PGE₂ stimulates the release of renin from the granular cells of afferent arterioles, especially during volume depletion. In the present study, newly established mouse macula densa cells (NE-MD) were used to directly determine whether COX-2 gene expression was regulated by low luminal [NaCl] concentration and other cellular signals, such as cAMP and Ca²⁺. Results. (1) Microarray analysis revealed that expression of COX-2 and EP4 mRNAs was increased 1.3- and 1.5-fold, respectively, in the presence of 12 μM furosemide (an inhibitor of Na⁺-K⁺-2Cl⁻ cotransporter). (2) Real-time RT-PCR showed a significant increase in COX-2 mRNA when NE-MD cells were incubated with low [Cl⁻] solution, furosemide or forskolin. Low [Cl⁻]-induced increase in COX-2 mRNA expression was completely reversed by either EIPA or BAPTA-AM (a membrane-permeable Ca²⁺ chelator). (3) RT-PCR revealed that NE-MD cells did not express EP1-EP3 receptors, but EP4 receptor. (4) Addition of PGE₂ to the medium elevated the intracellular cAMP concentration, which then resulted in the increase of COX-2 mRNA expression. (5) Treatment of NE-MD cells with low [Cl⁻] increased PGE₂ generation. Conclusions. These results suggest that COX-2 gene expression may be regulated by either extracellular [NaCl] or changes in the levels of intracellular cAMP, pH, and Ca²⁺. [NaCl] -inducible stimulation of COX-2 may influence the tubuloglomerular feedback by generating PGE₂. [J Physiol Sci. 2008;58 Suppl:S114]

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It remains unclear whether renal sympathetic nerve activity (RSNA) influences chronic regulation of renal sodium excretion. The present study was designed to examine the contribution of RSNA to long-term regulation of renal sodium excretion and sodium balance during changes in low, normal, and high sodium intake. Male Wistar rats were subjected either renal denervation (RD; n=9) or Sham surgery (n=17) and instrumented chronically with bipolar stainless steel electrodes for measurements of RSNA, electrocardiogram and electromyogram. The rats consumed very low-sodium pellet diet throughout and drank 4 different NaCl drinking solutions: 0 (low sodium), 50 (normal sodium), 154 (high sodium), and 308 (high sodium) mEq/L NaCl drinking solution. The experiment consisted of a 3 day control period (normal sodium), 3 days sodium unloading (low sodium)/loading (high sodium), and 3 days recovery period (normal sodium). During low sodium intake, cumulative sodium balance progressively decreased in both Sham and RS rats while the magnitude of decrease in the cumulative sodium balance in RD rats was greater than that in Sham rats. During high sodium intake, the magnitude of increase in the cumulative sodium balance in Sham rats was greater than that in RD rats. These data suggest that RSNA play a significant role in retaining sodium within the body during low sodium intake and also even during high sodium intake in rats. [J Physiol Sci. 2008;58 Suppl:S115]

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It is well known that aldosterone and osmotic stress are important physiological factors to regulate the epithelial Na⁺ channel (ENaC)-mediated Na⁺ reabsorption in renal epithelial cells. To investigate an interaction between aldosterone and hypotonic stress on stimulation of ENaC-mediated Na⁺ reabsorption in renal epithelial cells, we observed whether application of aldosterone (1 microM for 24h) modifies the action of hypotonic stress on the ENaC-mediated Na⁺ reabsorption by measuring the benzamil (a specific inhibitor for ENaC)-sensitive short-circuit current in renal epithelial A6 cells. The observations in the present study suggest that: (1) most ENaCs in cells untreated with aldosterone are translocated to Golgi apparatus, (2) major parts of aldosterone-generated ENaCs are located at the endoplasmic reticulum, (3) aldosterone diminishes the endocytosis rate of ENaCs from the apical membrane without any significant changes in the insertion rate of ENaCs into the apical membrane, and (4) addition of sucrose after hypotonic stress stimulates the endocytosis of ENaCs, and elongates the functional life time of ENaCs by enhancing recycle of ENaCs into the endoplasmic reticulum in a retrograde manner. This work was supported by Grants-in-Aid from Japan Society of The Promotion of Science (17390057, 17590191,18659056, and 19590212), Fuji Foundation for Protein Research, and The Salt Science Research Foundation (0736). [J Physiol Sci. 2008;58 Suppl:S115]

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In A6 cells, a renal cell line derived from Xenopus laevis, hypotonic stress stimulates Na⁺ transport. Hypotonic action on the Na⁺ transport is composed of two phases, a non-genomic early phase and a genomic delayed phase. Although previous works reported that during the genomic phase hypotonic stress stimulates the transcription of Na⁺ transport-related genes, such as serum- and glucocorticoid-inducible kinase 1 (SGK1) and subunits of epithelial sodium channel (ENaC), increasing the Na⁺ transport, the mechanism is still unknown. We focused the present study on the role of the intracellular Ca²⁺ in the hypotonicity-induced SGK1 and ENaC subunits transcription since hypotonic stress raises the intracellular Ca²⁺ concentration in A6 cells. In the present study, we observed that: 1) BAPTA/AM and W7 blunted the hypotonicity-induced expression of SGK1 mRNA and protein, 2) ionomycin dose-dependently stimulated expression of SGK1 mRNA and protein under an isotonic condition and the time course of the stimulatory effect of ionomycin on SGK1 mRNA was remarkably similar to that of hypotonic action on SGK1 mRNA, 3) hypotonic stress stimulated the transcription of three ENaC subunits in an intracellular Ca²⁺-dependent manner, and 4) BAPTA/AM retarded the delayed phase of hypotonic stress-induced Na⁺ transport without any effect on the early phase. These observations indicate for the first time that the intracellular Ca²⁺ plays a role as the second messenger in the hypotonic stress-induced Na⁺ transport by stimulating the transcription of SGK1 and ENaC subunits. [J Physiol Sci. 2008;58 Suppl:S115]

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To better understand the neuronal mechanism of the inspiratory pattern formation, we examined the spatiotemporal activity profile of phrenic motoneurons. We applied an voltage-imaging technique to newborn rat brainstem-spinal cord preparations. The preparations were placed in a recording chamber with a transversely cut plane of the spinal cord at the C4/C5 level horizontal. We recorded respiratory-related depolarizing optical signals on the transversely cut plane of the spinal cord simultaneously with the inspiratory discharge from the C4 ventral root using a high-speed optical recording system MiCAM01. We succeeded in visualizing the dynamic processes of the respiratory-related neuronal excitation propagation on the cut plane of the spinal cord. We found that the activity of phrenic motoneurons consisted of spatially distinct two subpopulations; the inspiratory activity first appeared in the ventrolateral portion of the ventral horn (VL) with a rapid onset and decrementing pattern starting simultaneously with the C4 ventral root discharge, and then another inspiratory activity appeared in the ventromedial portion of the ventral horn (VM). The activity in VL was larger and persisted longer than that in VM. We conclude that the phrenic motoneuron pool is composed of topographically and functionally different two subpopulations and they together generate the phrenic inspiratory discharge pattern. [J Physiol Sci. 2008;58 Suppl:S115]

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