Rationale) Seizure susceptibility and epileptogenesis depend not only on protein associated with glutamatergic and/or GABAergic neuronal transmission, but also on the redox. In glutamate transporter knockout (GLAST KO) mice, the kindling phenomena in GLAST KO developed more slowly while the after discharge duration (ADD) was briefer than that of the control C57BL-6J mice. To explain these phenomena, we focused the hippocampal redox state in the GLAST KO mice. Matreial and Methods) To measure anti-oxidant ability, the elimination of nitroxide radical was measured by usage of microdialysis combined with X-band ESR spectroscopy. Resluts) Nitroxide radical in the dialysate was eliminated exponentially, which means endogenous anti-oxidant reduced the paramagnetism of nitroxide radical. Half-life in GLAST KO. Discussion) Kindling-induced after discharge in electroencepharograms depends on the protein associated with glutamatergic and/or GABAergic neuronal transmission. But, there was no alteration except for increased GAT-3 expression, which supports enhanced GABAergic inhibition. In addition to enhanced GABAergic inhibition, Ca++ influx necessary for kindling development would be suppressed by oxidized redox state. From these physiological background, kindling development of GLAST KO mice was slower rather than taht of control. [J Physiol Sci. 2007;57 Suppl:S115]

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Bradykinin (BK) has been reported to be a mediator of brain damage in acute insults. Receptors for BK have been identified on microglia, the pathologic sensors of the brain, as well as in astrocytes. Here we report that BK attenuated lipopolysaccharide (LPS)-induced release of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL1-β) from glial cells, thus acting as an anti-inflammatory mediator in the brain. This effect was mimicked by raising intracellular cAMP or stimulating the prostanoid receptors EP2 and EP4, while it was abolished by a cAMP antagonist, a prostanoid receptor antagonist, or by an inhibitor of the inducible cyclooxygenase COX-2. BK also enhanced formation of prostaglandin E2, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) in glial cells. BK reduced LPS-induced neuronal death in neuron-glia co-cultures. This was probably mediated via glial cells since it did not affect TNF-α-induced neuronal death in pure neuronal cultures. Our data imply that BK has anti-inflammatory and neuroprotective effects in the central nervous system (CNS) by modulating glial function. [J Physiol Sci. 2007;57 Suppl:S115]

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Several lines of evidence have suggested that prostaglandin E₂ (PGE₂) is one of the intermediate messengers between the brain and immune system in, e.g., the infection-induced fever and endocrine responses. In the present study we investigated an involvement of PGE₂ in the immobilization (IMB, 1 hr)-induced suppression of splenic natural killer (NK) cell activity and its hypothalamo-sympathetic mechanisms. Intracerebroventricular (ICV) injection of a cyclooxygenase inhibitor, diclofenac (1 μg/1 μl saline, 15 min before IMB), partially blocked the IMB-induced suppression of NK activity in rats. In addition, diclofenac inhibited the IMB-induced expression of Fos protein in the hypothalamic paraventricular nucleus (PVN). An ICV injection (1 μg/l μl) of PGE₂ suppressed NK activity, while microinjection (0.1 μg/0.1 μl) study showed that the acting sites of PGE2 were the medical preoptic hypothalamus (MPO) and PVN, but not the ventromedial hypothalamus. Interestingly, microinjection of PGE₂ into the lateral hypothalamic area enhanced NK activity. We have shown that an activation of the splenic sympathetic nerve (SSN) induces a suppression of splenic NK activity, and ICV injection of PGE₂ increased SSN activity through EP₁ receptors. In accordance with these and present findings, microinjection of PGE₂ into the MPO and PVN, but not VMH, increased SSN activity, while the LHA injection decreased the SSN activity. These findings, taken together, suggest that hypothalamic PGE₂ plays a role in the stress-induced immunomodulation. [J Physiol Sci. 2007;57 Suppl:S115]

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The fetal brain is known to be survival from severe hypoxia during delivery, although its physiological mechanism remains unclear. We studied the roles of Bcl-2 (caspase-3 suppressor) and Bax (caspase-3 activator) in the hypoxia-induced activation of caspase-3 in the superior colliculus (SC) in a fetal rat, which was still connected with the anesthetized dam (urethane, 1.2-1.4 g/kg, i.p.) by the umbilical cord. Caspase-3 activity was measured with fluorescent caspase-3 substrate using an optical imaging system. Hypoxia was induced by occlusion of the umbilical cord for 5 min, and repeated every 1 hr up to 3 hrs after the initial occlusion. We observed a transient rise in caspase-3 activity following the initial occlusion. After reperfusion of the umbilical cord, caspase-3 activity gradually increased and afterward returned almost to the control level. Caspase-3 activation was more robust and long-lasting following the 3rd and 4th occlusion compared to the initial occlusion. Apoptosis of fetal SC cells was significantly increased after the umbilical occlusion. Application of Bcl-2 inhibitors unexpectedly suppressed the hypoxia-induced caspase-3 activation, while Bax inhibitors caused no effect. Further experiments are needed to clarify the unexpected result on Bcl-2 in association with apoptosis. [J Physiol Sci. 2007;57 Suppl:S115]

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Although large inter-individual differences in stress response are found in any animal or human population, the detailed mechanism is poorly understood. Significant differences are observed not only in the adequate responses to stressors, but also in the variability of stress-induced susceptibility. Understanding the causes of individual differences and their consequences in terms of adaptive capacity and vulnerability to disease is a major challenge of modern life sciences. In this study we compared hypothalamus transcriptional profiles between two different recovery patterns (fast recovery vs slow recovery) from restraint stress in rats using oligonucleotide microarray. Two recovery patterns were determined by the decline of plasma ACTH and corticosterone levels during one hour recovery period after stress. A real-time quantitative RT-PCR was applied to validate the differential expressed genes. It was found that talin, an important focal adhesion component that binds to multiple adhesion molecules including integrins, vinculin, focal adhesion kinase (FAK) and actin, was two fold up-regulated in fast recovery group. The results provided the first evidence that talin was involved in the recovery process from restraint stress in rats. As talin binding to integrin ß-tails is a final common step in integrin activation, further study will be needed to clarify the talin-mediated recovery mechanism after stress. [J Physiol Sci. 2007;57 Suppl:S116]

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It is known that the reactive oxygen species (ROS) affected several types of diseases including rheumatoid arthritis. We reported that moxibustion (MOX) applied to the Meimon (GV4) acupoint delayed the onset and suppressed symptoms in the collagen-induced arthritis (CIA) mice. Present study was undertaken to examine the effect of MOX on the production of ROS by measuring hydroperoxide content in plasma of CIA mice. DBA/1J mice were divided into four groups. CIA group: immunized with bovine typeII collagen, CIA+MOX group: immunized and applied moxibustion, with 1mg moxa cone 5 times a day to the Meimon(GV4) acupoint 3 times per week for 2 weeks, Control group: no immunization and no treatment, and Control+MOX group: applied moxbustion only. We evaluated the severity of arthritis by the arthritis score method and measured plasma hydroperoxide level by d-ROM test. Arthritis score was gradually increased from day28 and peaked on day35 in CIA and CIA+MOX group. The score in CIA+MOX group was significantly suppressed compared to CIA group. The level of hydroperoxide in CIA group was highest in all the groups on day28 and 35, while the level of hydroperoxide in CIA+MOX group was significantly lower than that in CIA group. These results suggested that moxibustion might suppress ROS production and severity of arthritis symptoms in CIA mice. [J Physiol Sci. 2007;57 Suppl:S116]

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Local gamma-ray irradiation is applied clinically (Gamma Knife^®) to functional neurological diseases such as epilepsy and trigeminal neuralgia. However, the neurophysiological mechanisms of such effects are not clear yet. The effects of gamma-ray irradiation on energy metabolism of rat brain were evaluated using ³¹P-nuclear magnetic resonance (NMR). Whole brains of Wistar rats were irradiated with a sub-necrotic dose (60Gy) of gamma-ray (Gammacell^® 40 Exactor, Nordian). One week after the irradiation, the brain slices (400 μm) were subject to ³¹P-NMR study (AMX300wb^®, Bruker). The slices were perfused with standard artificial cerebrospinal fluid bubbled with 95% O₂ +5% CO₂ in a test tube. Levels of high-energy phosphates, phosphocreatine (PCr) and γ-ATP, were measured after the high-K⁺ stress (60 mM) for 16 min, repeated with an interval of one hour. PCr and γ-ATP levels after the second high K⁺ stress were significantly lower in the gamma-ray irradiated rats than in the control rats (p<0.05), whereas there were no differences after the first stress. These results suggested that development of energetic tolerance to high K⁺ stress was reduced in gamma-irradiated brain. Such effects on energy metabolism of brain may influence the glutamate/glutamine cycle between neurons and astrocytes, hence neurotransmission was impaired. These mechanisms may play a role in the clinical application of gamma-ray to functional neurological diseases. [J Physiol Sci. 2007;57 Suppl:S116]

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Acute liver disease was induced in mice by carbon tetrachloride treatment. Levels of serum albumin, F-SH, T-SH, B-SH and total protein in the blood were then measured at 24 h after carbon tetrachloride intoxication.In comparison to controls, the liver injury group showed significantly greater increases in serum albumin (1.11-fold increase relative to controls, p<0.01), F-SH (1.48, p<0.005), B-SH (1.64, p<0.001) and T-SH (1.59, p<0.001).A correlation was observed between increases in B-SH and F-SH, thus suggesting that B-SH increases as a result of increased F-SH, with reactivity and subsequent binding of the products of liver injury. These findings suggest that F-SH acts as a buffer for substances, such as unnecessary or waste products in the body, and that a complex mechanism is involved in the production of SH groups. [J Physiol Sci. 2007;57 Suppl:S116]

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In patients with atrial septal defect (ASD), increased pulmonary blood flow by left-to-right shunting suppresses the respiratory vagal input to the heart and reduces the change in RR interval. We investigated the effect of increased shunt volume on the QT/RR relation and QT interval variability to mean the repolarization lability.The subjects were 32 patients with ASD. ECG was performed concurrent with echocardiography and the QT and RR intervals were determined. The QT/RR relation of 120 consecutive heart beats. In Doppler echocardiography, the pulmonary-to-systemic blood flow ratio was determined and the subjects were classified into the high- (2.0 or more) and low-shunt (less than 2.0) groups. In QT/RR linear regression and QT variability index (QTvi) were compared between the high- and low-shunt groups. Unpaired t-test for significance test was used.In the two groups, the high-shunt group exhibited a tendency toward variance, compared with the low-shunt group (p=0.007). In addition, QTvi were -0.856±0.632 and -0.091±0.718 in the low and high-shunt groups, respectively (p=0.0002).In ASD patients, reduced respiratory vagal input that was induced by an increased left-to-right shunt enhanced the QT/RR variance and increased QTvi. [J Physiol Sci. 2007;57 Suppl:S117]

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Microglial cell has been demonstrated to be involved in various diseases, such as Alzheimer, Parkinson diseases and multiple sclerosis. In the previous study we demonstrated that an exposure of rats to acute stress induced rapid morphological microglial activation. However, the stress-induced microglial activation was not accompanied with the induction of proinflammatory markers, IL-1β, IL-6, and iNOS. In the present study, we sought to determine the effect of adrenalectomy on the microglial activation as well as the induction of proinflammatory markers. We exposed the rats, sham-operated and adrenalectomized (ADX) rats, to restraint combined with water immersion stress for 2 hours. Immunohistochemistry with OX-42 demonstrated that morphological microglial activation is enhanced in the ADX rats as compared to that in sham-operated rats. Following the exposures to acute stress, the morphological microglial activation was intensified in the ADX-rats than sham-operated rats. Importantly, the induction of proinflammatory markers, such as IL-1β, IL-6 and iNOS, was observed only in the ADX-rats, but not in sham-operated rats, in acute stress. In addition, the morphological microglial activation as well as the production of proinflammatory cytokine markers was significantly suppressed by the treatments with glucocorticoids. Finally, double immunohistochemistry clearly demonstrated the co-localization of those inflammatory markers in the activated microglial cells. Thus, the present study suggests that glucocorticoids may have suppressive effects on the microglial activation as well as the induction of proinflammatory markers. [J Physiol Sci. 2007;57 Suppl:S117]

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"Ca²⁺ paradox" is the phenomenon whereby the intracellular concentration of Ca²⁺ paradoxically increases during reperfusion with normal Ca²⁺-containing media after brief exposure to a Ca²⁺-free solution. We found that prior exposure of the cultured astrocytes to a low Ca²⁺ solution for 60 min significantly decreased their intracellular ATP level ([ATP]_i) and injured them after reperfusion with a normal Ca²⁺ medium for 72 h. The present study aims at elucidating what mechanisms are involved in this ATP reduction leading to cell injury. In our previous study, we revealed that the intracellular concentration of Na⁺ ([Na⁺]_i) in astrocytes increased significantly during the Ca²⁺-free treatment. Thus, we first thought that this elevation of [Na⁺]_i increased the function of Na⁺/K⁺ ATPase, leading to the increase of ATP consumption. However, ouabain treatment didn't significantly change their [ATP]_i. Previous studies have revealed that Ca²⁺-free or low Ca²⁺ treatment induces the release of ATP to the extracellular space via either connexin hemi-channels or anion channels. When cultures were treated with either FFA or heptanol, inhibitors of these channels, the rise in extracellular ATP ([ATP]_o) was reduced, without maintaining intracellular amount of ATP. Both the ATP release and the [ATP]_i reduction were significantly attenuated by treatment with suramin, an inhibitor of P2Y receptors. These results suggested that astrocytic ATP release during Ca²⁺-free treatment induced their fall in [ATP]_i via the activation of P2Y1 receptor. [J Physiol Sci. 2007;57 Suppl:S117]

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To investigate a possible correlation between Epstein-Barr virus (EBV) infection statuses including its latent gene expression and expression of allergy-related genes in human tonsil-derived cells. In the tonsil derived cells from the patients undergoing routine tonsillectomies for palatine tonsil hypertrophy or tonsillar focal infection, the presence of EBV DNA and mRNA expressions of latent membrane protein (LMP)-1, Cε chain, and activation induced cytidine deaminase (AID) were detected by PCR and RT-PCR analysis, respectively. Of all the twelve patients, PCR products amplified from EBV DNA BamHI W fragment were detected in the tonsils from the 10 patients (83.3%). LMP1 mRNA expressions were confirmed in the 6 patients (50%). Both LMP1 mRNA expressions and EBV DNA were detected in the 5 patients. EBV DNA, but not LMP1 mRNA expression, was detected in the 5 patients. LMP1 mRNA expression, but not EBV DNA, was detected in one patient. In another patient, neither EBV DNA nor LMP1 mRNA expression was confirmed. Cε mRNA expressions were confirmed in all the 12 patients along with AID mRNA expressions. The Fishers exact probability test revealed a statistically significant correlation between LMP1 and Cε gene expressions, indicating that Cε mRNA expression level is significantly higher in the LMP1 positive samples than in the negative samples. Thus, EVB infection is a notable factor capable of exacerbating allergic inflammation. [J Physiol Sci. 2007;57 Suppl:S117]

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Daidzein (Da), a major soy isoflavone, had been shown to induce differentiation in the murine neuroblastoma Neuro-2a (BU-1) cells mediated by estrogen receptor, and other health effects, such as reduction of tumour invasivenss and immunomodulatory effect. In this study, we compared the effects and signaling mechanisms of differentiation inducers (DIs) Da, all-trans retinoic acid (ATRA) and prostaglandin E2 (PGE2), in BU-1 cells. Da, ATRA, and PGE2 could inhibit the growth of BU-1 cells. Cell cycle analysis indicated that Da reduced cells at the G2/M phase of the cell cycle; while both ATRA and PGE2 caused a G2/M phase arrest. Cell death studies found that Da but not ATRA or PGE2 induced apoptosis; where PGE2 exerted anti-apoptotic effect in the cells. All the DIs triggered neurite outgrowth; however, only Da and ATRA could elicit functional differentiation as indicated by elevated acetylcholine esterase activity. The crosstalk of cellular response signaling was probed by the inhibition of MAPK pathways. The inhibition of p38 MAPK and ERK pathways suppressed the growth inhibition induced by ATRA: p38 MAPK inhibition was coupled with cell differentiation and ERK inhibition was linked to cell cycle modulation. In PGE2-treated cells, the inhibition of ERK pathway potentiated the growth inhibition, reduced cells at the S phase, and suppressed the anti-apoptotic effect of PGE2. The effects Da were independent of MAPK activities. Our results suggested that daidzein has distinct properties from other DIs, which might pose some novel therapeutic possibilities. [J Physiol Sci. 2007;57 Suppl:S118]

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It has been well established that intra- and extracellular mechanical environments affect development of focal adhesions (FAs), however, little is known about its molecular mechanisms. FA is a site where actin polymerizes, and the assembly of produced F-actin at FAs is thought to be crucial for FA development. Therefore, certain actin-modulating proteins at FAs may be involved in the mechanical regulation of FA development. In this study, we focused on zyxin, an FA-associated protein having actin polymerizing capability, as a candidate molecule and examined its distribution in response to changes in the mechanical environment in cultured fibloblasts. When cell contractility was inhibited by the myosin II inhibitor, blebbistatin, zyxin dislocated from FAs, which was the earliest remarkable event we could notice. This result suggested that the zyxin assembly at FAs is regulated by the forces applied to FAs. To confirm this idea, we applied uniaxial 50%-stretch to the blebbistatin-treated cells and found that zyxin was reaccumulated at FAs. When fluorescent-labeled G-actin was introduced into permeabilized cells, G-actin was incorporated at zyxin-positive FAs but not at zyxin-negative ones. Incorporation of G-actin at FAs was not observed in the blebbistatin-treated cells, while application of uniaxial stretch restored the G-actin incorporation. These results suggest that zyxin is associated with certain mechano-sensitive machinery at FAs and may regulate actin polymerization at FAs in a force-dependent manner. [J Physiol Sci. 2007;57 Suppl:S118]

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Cells expressing NG2 chondroitin sulfate proteoglycan (NG2) in the brain have been considered to be oligodendrocyte progenitor cells. We found, however, most cells expressing NG2 also expressed Iba1, a microglia/macrophage marker, in the core of ischemic brain lesions of rats that were subjected to transient middle cerebral artery occlusion (MCAO). The cells expressing both Iba1 and NG2 (Brain Iba1+/NG2+ Cells; BINCs) displayed macrophage-like morphology and had phagocytic activity. BINCs were highly proliferative and expressed another OPC marker platelet-derived growth factor α receptor (PDGFαR). BINCs isolated from the core of MCAO lesions proliferated in response to PDGF-AA, a ligand for PDGFαR. To label dividing cells in the ischemic core of the MCAO lesion at 2 days post-reperfusion (2 dpr), 5-bromo-2-deoxyuridine (BrdU) was administered to the ischemic rats. Then, about 80% of BrdU-labeled cells were BINCs. Based on this result, BINCs were killed by administrating 5-fluorouracil (5FU) to rats at 2 dpr. The administration of 5FU increased apparently infarct volume that accompanied marked decrease of BINCs in number, and nearly 60% of rats died by 14 dpr. These results suggest that BINCs played favorable roles in ischemic lesions to reduce the infarct volumes. Isolated BINCs can transdifferentiate into cells with neuroectodermal phenotypes and this multipotentiality might be involved in the regeneration of the ischemic brain. [J Physiol Sci. 2007;57 Suppl:S118]

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Background: A new PKA independent signaling pathway of cAMP was identified by the discovery of "Epacs", of which the two isoforms, Epac1 and Epac2, exist.Objective: The aim of this study was to examine the effect of Epac1 and Epac2 on migration in human melanoma cells. Methods: Migration of two human melanoma cell lines, namely SK-MEL-2 (MEL-2) and SK-MEL-24 (MEL-24) was analyzed using modified Boyden-chamber method. Overexpression of Epac1 and Epac2 proteins were obtained by adeno-virus infection.Results: The epac specific cAMP analogue 8-pCPT-2-O-Me-cAMP (8-pCPT) enhanced cell migration in MEL-2 by 41% (n=2). Epac1 over-expression increased cell migration in MEL-2 and MEL-24 11.3 fold (P<0.01; n=4) and 1.6 fold (P<0.05; n=7), respectively. This increase was further enhanced 2.2 fold (P<0.001) by cAMP analogue in ME L-2, whereas in MEL-24 further increase was not observed. Augmentation of migration was observed in Epac2 overexpressed cells by 3 fold in MEL-2 without any additional effect of 8-pCPT. These Epac induced enhancements were suppressed by PI3-kinase antagonist wortmannin in a dose dependent manner.Conclusion: Our study show that both Epac overexpression and stimulation with Epac specific cAMP analogue enhanced migration in human melanoma cells with different efficacy in comparison of MEL-2 and MEL-24 cells. These results suggest the involvement of Epac pathway in migration of melanoma cells, which includes the activation of PI3-kinase, and also that the degree of involvement is different among various melanoma cell types. [J Physiol Sci. 2007;57 Suppl:S119]

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The caveolin family of the membrane anchoring proteins accumulates various growth receptors in caveolae and inhibits their function. Here, we identify caveolin as a potent enhancer of insulin signal when overexpressed in the liver in vivo, where endogenous caveolin is little expressed. Adenovirus-mediated caveolin-3 gene transfer to the liver led to a marked increase in hepatic glycogen synthesis in diabetic mice (p<0.05, n=4), but not in non-diabetic lean mice, and was accompanied by a decrease in mRNA expression of phosphoenlopyruvate carboxykinase (p<0.05, n=5) and an increase in that of glucokinase (p<0.05, n=5). There was a marked increase in insulin sensitivity in diabetic obese mice as exemplified by decreased fasting blood glucose levels (p<0.05, n=8) and improved glucose tolerant test performance. These effects were attributed mostly to increased insulin receptor activity and caveolin-mediated, direct inhibition of PTP1B activity (p<0.05, n=4), of which expression was significantly increased in obese mouse liver (p<0.05, n=4). Overexpression of caveolin-3 in hepatic cells enhanced the insulin signal as well. Our results suggest that caveolin-3 is an important, endogenous regulator of glucose metabolism that can enhance insulin signal, in particular, under pathological conditions where phosphatase activity is upregulated. [J Physiol Sci. 2007;57 Suppl:S119]

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Bionanocapsules (BNCs) are hollow nanoparticles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNC is useful for delivering genes, siRNAs and drugs to the human liver as drug delivery system (DDS) carrier (Nat Biotech 21, 885 (2003)). Brain tumor is difficult to be extirpated widely and liable to recurrence because a brain is an extremely important part. Although a precise radiotherapy using three-dimensional image diagnosis such as gamma knife has proceeded, the recurrence rate is still high. Therefore, it is important to develop DDS targeting to brain tumor.In the present study, we developed a novel DDS to brain tumor using BNCs, which selectively targeted to brain tumor. Epidermal growth factor receptor (EGFR) is overexpressed in human glioma. The pre-S1 peptide displays on the surface of BNC as the specific ligand for receptor binding on human hepatocytes. We replaced the pre-S1 peptide with antibody affinity motif of proteinA and made hybrid BNC binding anti-human EGFR antibody. The hybrid BNCs were efficiently delivered in glioma cells but not normal glial cells. Moreover, we confirmed the specific delivery of the hybrid BNC to brain tumor in vivo model of brain tumor. These results suggest that this new approach using BNCs provides a promising system for brain tumor-targeted drug delivery. [J Physiol Sci. 2007;57 Suppl:S119]

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Objective: The atomic force microscope (AFM) is a useful tool for studying proteins at the single molecule level, however, these studies in physiologically relevant conditions have been restricted, in part, by both the noise and speed limitations of the AFM. Recent progress in AFM with small cantilever enables us to observe individual protein interactions in faster and quieter conditions. Here we have observed, in real time, submembranous structures of the cytoplasmic side of plasma membrane of human umbilical vein cord endothelial cells (HUVECs). Methods: HUVECs cultured on quartz specimen holders were sonicated in hypotonic solution, then attached on the scanner of a high-speed AFM (NVB500, Olympus). The cantilevers used were typically 10 μm long, 2 μm wide, and 100 nm thick. Each cantilever had an electron beam deposited tip that was 600-800 nm long. All imaging was done in tapping mode to minimize sample damage and performed at ambient temperature. Results and Discussion: The cytosplasmic face of the fixed basal HUVEC plasma membrane showed meshwork structure and particles of various size (10–40 nm) at lower magnification (800 x 600 nm). Single actin filaments formed three-dimensional lattice at higher magnification (200 x 150 nm). These observations are consistent with our previous observation by confocal laser microscope and quick-freeze deep-etch electron microscope. The high-speed AFM will open the possibility of studying membrane proteins such as ion channels at single molecule level. [J Physiol Sci. 2007;57 Suppl:S119]

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The apical membrane of renal cortical collecting duct (CCD) faces the tubular fluid whose osmolarity is relatively low. Using the patch-clamp technique, we investigated the effect of reducing extracellular osmolarity on ion conductance of the apical membrane of principal cells in freshly isolated rat CCD. In cell-attached patches, three distinct types of the ion conductance formed by 30 pS K⁺, 10 pS Cl⁻ and 190 pS K⁺ channels were observed. Although the open probability (NP_o) of the former two channels was relatively high, that of the 190 pS large K⁺ channel was extremely low under the control condition. NP_o of the large K⁺ channel was raised by cytosoic free Ca²⁺ and depolarization. The exposure of the cells to hypotonic solution did not significantly influence NP_o of 30 pS K⁺ and 10 pS Cl⁻ channels, while NP_o of the large K⁺ channel was markedly enhanced within 5 min after the exposure to the hypotonic solution. The hypotonic solution-induced increase of NP_o of the large K⁺ channel was not observed in the absence of extracellular Ca²⁺ . Fluorescence imaging of intracellular Ca²⁺ concentration ([Ca²⁺]_i) with fura-2 showed that the hypotonic solution evoked transient increase in [Ca²⁺]_i, but did not increase [Ca²⁺]_i when the extracellular Ca²⁺ was removed. These results indicate that reducing extracellular osmolarity induces Ca²⁺-dependent activation of the large K⁺ channel due to an increase in [Ca²⁺]_i by a Ca²⁺ entry from the extracellular source in rat CCD. [J Physiol Sci. 2007;57 Suppl:S120]

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Ion channels present in the apical membrane of cultured mouse renal collecting tubule (M-1) principal cells were investigated using the patch-clamp technique. Two types of channel current, small current with high open probability (Po) and intermediate current with low Po, were observed in the apical surface of confluent mono-layered M-1 principal cells in cell-attached patches under the control condition. It was revealed that the small current was formed by inwardly rectifying small conductance (20–30 pS) K⁺ (SK) channels, and the intermediate current was formed by non-selective cation (NSC) channels with the conductance of about 40 pS. Addition of 8-BrcAMP (100 μM) to the bath solution enhanced activity of the SK channel, whereas that of KT5720 (500 nM), an inhibitor of protein kinase A (PKA), suppressed it in cell-attached patches. However, neither 8-BrcAMP nor KT5720 induced appreciable change in activity of the NSC channel. In inside-out patches, the SK channel required 1 mM MgATP in the cytoplasmic surface to maintain its activity, while the NSC channel was activated by cytoplasmic Ca²⁺. From these basic properties, it is suggested that the SK channel is an ATP-regulated channel which is subject to ROMK channel family, although the molecular identification of the SK channel as well as that of the NSC channel remains to be investigated. Further studies will be necessary to clarify the physiological roles of these ion channels in the function of mouse kidney collecting tubule. [J Physiol Sci. 2007;57 Suppl:S120]

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Salivary stone is thought to be a calcareous degenerative product occurred in the submandibular gland and parotid gland. The exact mechanism of the formation of calculi is not clear. By using a Raman spectroscopy, we studied the spectroscopic characterization of human salivary stone, and compared with that of the model materials such as enamel, dentine, bone tissues and synthetic hydroxyapatite (sHAP). Twelve human salivary stones removed by operations were used and its cross-section surfaces were investigated by a Raman spectroscopy. Enamel and dentin of impacted lower third molar tissues, the compact bone of rat femur and sHAP were measured as the model materials. Raman spectra were obtained by a JEOL JRS-FT6500N Raman spectrometer. A 1064 nm line from a continuous wave Nd:YAG laser was used for excitation. Raman spectral pattern for the human salivary stones was quite similar to that for enamel and sHAP, indicating that the principal constituent of the stone may be calcium phosphate and its structural characterization may be same as that for HAP. However, the spectral patterns for protein and CO₃²⁻ were also observed in some cases, suggesting that some protein and CO₃²⁻ may be one of the compositions of the stone. From these results, human salivary stones could be classified into three groups by the structural features; the HAP-structure group, the protein-rich HAP-structure group and the CO₃²⁻-rich HAP-structure group. [J Physiol Sci. 2007;57 Suppl:S120]

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Equivalent cross-relaxation rate (ECR) imaging (ECRI) is a measurement method that can be used to quantitatively evaluate a change in the protein-water interaction by MRI. The aim of this study is to evaluate utility of ECRI as an in vivo molecular imaging for living tissue such as lymph node. Fifteen patients with histologically confirmed invasive ductal carcinomas of the breast were studied. We adopted the off-resonance technique for preferential saturation of the immobile protons to evaluate the ECR values. The single saturation transfer pulse frequency was employed at the frequency 5 ppm downfield from the water resonance. The ECR value was defined as the percentage of single loss between unsaturated and saturated images. The ECRI was constructed on the basis of the percentage of ECR. The normal lymph node organization showed there were a large number of the cell and higher ECR value. On the other hand, the lymph nodes with metastases showed a small amount of the cell and a lower ECR value, and adipose tissue was almost no cells and the lowest ECR value. A statistically significant difference was observed in the ECR values of each tissue, and both numerical values were present on the first correlation line. In axillary lymph nodes with or without metastases, ECR value was well related to the number of the cell. Therefore, ECRI might be a potentially useful method for molecular imaging in tissue characterization of the lymph nodes. [J Physiol Sci. 2007;57 Suppl:S120]

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Genetically encoded fluorescent calcium indicator proteins are useful tools to study calcium dynamics in cellular activities. We developed and reported properties of a novel fluorescence resonance energy transfer (FRET)-based calcium indicator protein, designated F2C. F2C has cyan- and yellow- fluorescent proteins linked with a calpain sensitive sequence, and membrane-associated sequence in N-terminus. Primary cultures of rat Purkinje cells were transiently infected with Sindbis virus encoded this protein and fluorescent image analysis was performed. The fluorescence signal from F2C was repeatedly changed when intracellular calcium concentration was altered.Here, we report the generation of transgenic mice expressing F2C under control of tetracycline regulation system and analysis of these transgenic mice. F2C expression in transgenic mice was regulated by tetracycline repressor protein (TetR). We also generated transgenic mice which express TetR under the control of HCN4 promoter. F2C transgenic mice were crossbred with HCN4 transgenic mice to generate F2C-expressing mice and two lines of these crossbreeding mice expressed F2C in olfactory receptor neuron. F2C showed over 15% ratio changes by KCl application in olfactory bulb slices of these mice. Furthermore, in vivo experiment, F2C showed over 10% ratio changes by ATP application. [J Physiol Sci. 2007;57 Suppl:S121]

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UT-B1 is one of the isoforms of facilitated urea transporters that mediate rapid, passive movement of urea across cell membranes, and are candidates for regulators of urea permeability in the cell membrane. Our experimental results indicated the UT-B1 function as active water transporter against osmotic gradient in C6 glial cells. Exposure of C6 cells to a hyperosmotic (+ 300 mOsm/kg H₂O) solution containing glycerol or sucrose produced cell shrinkage (about 20% decrease) due to water efflux according to osmotic gradient for water movement. On the other hand, C6 cells showed cell swelling (about 6% increase) against osmotic gradient for water movement just after exposure to a hyperosmotic solution containing urea, indicating that water influx against osmotic gradient for water movement is accelerated by urea; i.e., urea performs active water transport. Exposure of C6 cells to hypertonic urea media with a specific inhibitor for UT-B, pCMBS (500 μM), blocked the urea-induced swelling, but induced immediate, abrupt cell shrinkage (about 27% decrease). The urea-induced cell swelling was significantly suppressed in the siRNA-induced UT-B1-knockdown C6 cells. Taken together, these observations indicate that UT-B1 acts as an active water transporter and as a key molecule of water and urea excretion through the so-called astrocyte network. [J Physiol Sci. 2007;57 Suppl:S121]

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Dynamic organization of cytoskeletons, such as microtubules, actin filaments and intermediate filaments, is crucial for the neurite outgrowth in neuronal cells. On the other hand, it is suggested that changes of intracellular chloride ion concentration is involved in regulation of various cellular functions such as cell cycle, proliferation, adhesion, migration, and dynamics of cytoskeleton. Neuronal cells are known to cooperate the K⁺/Cl⁻ cotransporter (KCC) and/or the Cl⁻-ATPase excreting Cl⁻ from the cells, and to incorporate Cl⁻ into the cells via the Na⁺/K⁺/2Cl⁻ cotransporter (NKCC). Further, it is also reported that the NKCC-like activity is up-regulated by NGF treatment in PC12 cells. In this study, we investigated the role of NKCC1 in NGF-induced neurite outgrowth in PC12D cells (a subclone of PC12 cells). Bumetanide, a specific inhibitor of NKCC, significantly inhibited the NGF-induced neurite outgrowth in PC12D cells. A similar phenomenon was observed in PC12D cells exposed to a low Cl⁻ concentration medium. Expression of NKCC1 protein was up-regulated by NGF treatment. Furthermore, knock down of NKCC1 by RNAi drastically diminished the NGF-induced neurite outgrowth. A study of GFP-tagged rNKCC1 transfection into PC12D for clarification of the cellular localization of NKCC1 indicated that the GFP-rNKCC1 was localized in growth cone during neurite outgrowth. Based upon these findings, we conclude that Cl⁻ uptake by NKCC1 localized in the growth cone promotes neurite outgrowth. [J Physiol Sci. 2007;57 Suppl:S121]

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Serum- and glucocorticoid-inducible kinase 1 (SGK1) plays an important role in the body NaCl homeostasis by fine tuning of renal NaCl reabsorption in the distal nephron via activity modulation, transcription, and trafficking of epithelial Na⁺ channel (ENaC). Although the SGK1 induction at mRNA and protein levels is involved in the stimulatory action of hypotonicity on Na⁺ transport, it is unknown how hypotonicity regulates the SGK1 expression. In the present study, we studied if the Ca²⁺ signal is involved in the hypotonic action on SGK1 expression and Na⁺ transport. Using QRT-PCR and western blotting, we observed that BAPTA/AM (an intracellular Ca²⁺ chelator) and W7 (a calmodulin antagonist) diminished the hypotonic induction of SGK1 mRNA and protein. Ionomycin (a Ca²⁺ ionophore) stimulated SGK1 transcription under an isotonic condition. In short-circuit current measurement, BAPTA/AM showed an inhibitory effect on Na⁺ current 60 min after hypotonic challenge, the time course of which correlated with that of hypotonic induction of SGK1 protein, suggesting the contribution of SGK1 to the hypotonicity-provoked Na⁺ transport. Regarding ENaC expression, a possible correlation was found between SGK1 activity and expression of αENaC, but not β or γENaC. Taken together, we conclude that hypotonic stress has genomic action on SGK1 mRNA expression in a Ca²⁺/calmodulin-dependent manner, stimulating Na⁺ transport and αENaC expression. JSPS17590191, 17390057 [J Physiol Sci. 2007;57 Suppl:S121]

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S1P has a critical role in vascular maturation in vivo during mammalian development. However, little is known about the therapeutic effect of S1P in ischemia-induced angiogenesis in adults. We investigated the effect of exogeneous S1P on angiogenesis in ischemic skeletal muscle in adult mice. Unilateral murine hindlimb ischemic model is an well-established in vivo angiogenesis assay system. We evaluated post-ischemic angiogenesis by monitoring post-surgical blood flow recovery with a laser doppler imager and the capillary density with anti-CD31 immunohistochemistry. First, we injected S1P solution into the ischemic muscle everyday during 28 days after surgery in C57BL6/J mice. The limb blood flow was significantly elevated between 10 and 28 days after surgery in S1P-injected mice compared to vehicle-injected mice. The capillary density was significantly increased in the S1P-administered group at 10 days after surgery. Next, we administrated several different doses of S1P into the ischemic lesion everyday. S1P accelerated recovery of the blood flow from ischemia and induced an increase in the capillary density in a dose-dependent manner. bFGF is known to be one of the most potent angiogenic factors, daily injection of bFGF increased the blood flow and the capillary density in the ischemic limb. The comparison of the effects between S1P and bFGF showed that S1P stimulated ischemia-induced angiogenesis to the similar extent of bFGF. These results suggest the possibility that S1P could be a potential therapeutic agent for ischemic diseases. [J Physiol Sci. 2007;57 Suppl:S122]

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Flavins are very versatile coenzymes, and flavoenzymes catalyze various oxidation-reduction reactions. The selection of a specific catalytic reaction from various functions is accomplished by the interactive network of flavin ring, apoenzyme, and substrate. Therefore, the information about the interaction is essential for elucidation of the reaction mechanism on molecular levels. We investigated the molecular interaction of FAD encountered in the active site of flavoproteins (medium-chain acyl-CoA dehydrogenase (MCAD) and electron-transferring flavoprotein (ETF)) by FTIR spectroscopy using C=O stretching as a probe. The 1645-cm⁻¹ band of FAD shifted to 1630 cm⁻¹ in the MCAD-bound form. The low frequency shift can be explained by hydrogen bonds at C(2)=O of the flavin in the MCAD active site. The band was observed at 1607 cm⁻¹ in the charge-transfer complex of MCAD with 3-thiaoctanoyl-CoA. In the case of ETF, two bands of C(4)=O stretching mode were observed at 1712 and 1686 cm⁻¹, indicating multiple conformations of ETF: one with a strong hydrogen-bond, and the other with a weak hydrogen-bond at C(4)=O. [J Physiol Sci. 2007;57 Suppl:S122]

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The purified electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii has one tightly bound FAD molecule as the cofactor and binds another FAD molecule added exogenously. Thus holo-ETF contains two FAD molecules. Here the FAD contained in purified ETF is named FAD-1 and the additionally bound FAD is named FAD-2. The function of ETF is receiving electrons from NADH and donating them to other flavoenyzmes, enoyl-CoA reductase and D-lactate dehydrogenase. When purified ETF is mixed with NADH, FAD-1 is reduced. When holo-ETF is mixed with NADH, both FAD-1 and FAD2 are reduced. These findings clarifies that FAD-1 directly receives electrons from NADH. However, it is unclear whether FAD-2 receives electrons from NADH or FAD-1. The reduction of FAD-1 and FAD-2 by NADH was too fast to investigate kinetically even with stopped flow apparatus. In this study, we used 8-CN-FAD, which has more positive standard redox potential than FAD. The holo-ETF whose FAD-1 was exchanged for 8-CN-FAD was prepared. If FAD-2 receives electrons from FAD-1, the reduction of FAD-2 should be perturbed by the exchange. When this holo-ETF was mixed with NADH, fully reduced 8-CN-FAD and one electron-reduced FAD-2 were formed in the dead time of the measurement, followed by slow additional one-electron reduction of FAD-2. This result indicates that FAD-2 does not receive electrons from NADH but from FAD-1, because the electron transfer from NADH must be simultaneous two-electron transfer. [J Physiol Sci. 2007;57 Suppl:S122]

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Polychlorinated biphenyls (PCBs) are well known pollutants that persist ubiquitously in the environment at low concentration. Some of their congeners may affect neuronal development and function by altering signal transduction and gene expression. However, the molecular mechanisms underlying their neurotoxicity are still not clear. Since Ca²⁺ is used as an intracellular signaling molecule, we investigated whether PCB alters Ca²⁺ oscillation in PC12 cells. We also analyzed the effect of PCB on membrane potential, since the alteration of Ca²⁺ oscillation is often associated with its changes.PC12 cells were loaded with Fura-2 AM for Ca²⁺ oscillation and DiBAC₄(3) for membrane potential. PCB (4(OH)-2',3,3',4',5'-penta CB, OH-PCB) was added to the medium at a final concentration of 10⁻⁸ M. Fluorescence of Fura-2 and DiBAC₄(3) was monitored using a CCD camera.Exposure to OH-PCB increased the intracellular Ca²⁺ content up to 3 times. The rise was gradual starting right after the PCB treatment. Interestingly, the increase in intracellular Ca²⁺ preceded the plasma membrane depolarization. Membrane potential showed an increase of 6 times in average with a delay of 1 to 2 min after application of PCB. Deprivation of extracellular Ca²⁺ reduced significantly the increase in cytoplasmic Ca²⁺ after OH-PCB treatment. Our results indicate that low-dose OH-PCBs alter membrane potential as well as disrupt Ca²⁺ homeostasis in part by inducing extracellular Ca²⁺ influx. These mechanisms may partly mediate its toxicity. [J Physiol Sci. 2007;57 Suppl:S122]

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Sympathetic stimulation induces secretion of saliva of which volume is smaller than that by parasympathetic stimulation. However, the cellular mechanism is unclear. Since salivary fluid secretion depends on Cl⁻ secretion from acini of salivary glands, we investigated Cl⁻ secretion induced by the sympathetic transmitter, norepinephrine (NE), adopting the gramicidin-perforated patch recording method to measure Cl⁻ secretion as an anion current at the K⁺ equilibrium potential, -80 mV, in acinar cells. NE induced an oscillatory anion current of which amplitude gradually decreased to a steady-state level in submandibular acinar cells. The β-aderenergic antagonist, propranolol, did not induce any current, but inhibited the gradual decrease in the NE-induced current, suggesting that the current is induced via the α-adrenergic stimulation by NE and suppressed via the β-adrenergic stimulation by NE and that the relatively small volume of saliva induced by sympathetic stimulation may be at least partially due to the reduction of Cl⁻ secretion in the acinar cells by the β-adrenergic stimulation. [J Physiol Sci. 2007;57 Suppl:S123]

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= Purpose = The physiological properties of monosaccharides have not been studied well because most of them are rare in the nature. Angiogenesis, formation of new capillary blood vessels, plays an important role in a variety of normal and abnormal physiological processes, such as in embryonic development, tumor growth and metastasis. In 2005, we reported the effects of 36 monosaccharides on the proliferation and the tube formation of human umbilical vein endothelial cells(HUVEC). Some of them inhibited the proliferation and/or the tube formation, and these active sugars possessed some common structures. In this work, we added the data of other 7 monosaccharides. Together with the previous data, further structure activity relationship will be discussed. = Conclusion = We previously reported that the absence of OH group at the equatorial position of C2 or C3 is essential to cause inhibition. This means the increase in the hydrophobicity or the decrease in the steric hindrance around the area. Present data also supported this structure activity relationship, and revealed that the former reason is responsible for the inhibition. [J Physiol Sci. 2007;57 Suppl:S123]

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To understand the biochemical basis for the function of the single subunit NADH-quinone (Q) oxidoreductase (Ndi1), we have overexpressed mature Ndi1 in Escherichia coli membranes. The Ndi1 purified from the membranes contained one FAD and showed enzymatic activities comparable to the original Ndi1 isolated from Saccharomyces cerevisiae. When extracted with Triton X-100, the isolated Ndi1 did not contain Q. The Q-bound form was easily reconstituted by incubation of the Q-free Ndi1 enzyme with ubiquinone-6. We compared the properties of Q-bound Ndi1 enzyme with those of Q-free Ndi1 enzyme, with higher activity found in the Q-bound enzyme. Although both are inhibited by the same concentration of AC0-11, the inhibitory mode of AC0-11 on Q-bound Ndi1 was distinct from that of Q-free Ndi1. When Ndi1 was incorporated into bovine heart submitochondrial particles (SMP), the Q-bound form, but not the Q-free form, established the NADH-linked respiratory activity which was insensitive to piericidin A but inhibited by KCN. Furthermore, Ndi1 produces H₂O₂ as isolated regardless of the presence of bound Q, and this H₂O₂ was eliminated when the Q-bound Ndi1, but not the Q-free Ndi1, was incorporated into SMP. The data suggest that the bound Q site is different from a catalytic site for Q and that Ndi1 in mitochondria bears the bound Q. Now, we are trying to identify the Q binding sites by site-directed mutagenesis technique. [J Physiol Sci. 2007;57 Suppl:S123]

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D-Allose, the C-3 epimer of D-glucose, is one of the rare sugars which are rare in nature. D-Allose was shown to have an inhibitory effect on cell proliferation of various human cancer cells. In particular, various lymphoma cell lines have been elucidated to have different susceptibility to D-allose. In other words, the proliferation of some human leukemia cell lines was significantly inhibited by D-allose, and some were not. In order to explain the different susceptibility, we chose 5 leukemia cell lines, MOLT-4F (T-cell lymphoblastic leukemia), IM-9 (bone marrow), HL-60 (acute promyelocytic leukemia), BALL-1 (acute lymphoblastoid leukemia) and Daudi cell (Burkitt lymphoma). Using these four cell lines, we measured the uptake of D-allose together with other monosaccharides using ₁₄C-labeled D-glucose, D-allose, D-fructose and D-psicose. We also investigated the expression pattern of all glucose transporters (GLUT) in these cells by the real-time reverse transcription-PCR (RT-PCR) method, since GLUTs are thought to be involved in uptake of monosaccharides including D-allose. In addition to the above analyses, we analyzed the expression of several cell-cycle related proteins using Western blotting. We discuss possible mechanisms which may explain the different susceptibility to D-allose-induced inhibition of cell proliferation among various leukemia cell lines. [J Physiol Sci. 2007;57 Suppl:S123]

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The volume-sensitive outwardly rectifying Cl⁻ channel (VSOR) is known to be involved in cell volume regulation after osmotic swelling called regulatory volume decrease (RVD), cell proliferation, cell cycle, and cell death. However, neither its molecular identity nor its activation mechanism is unknown. Since both VSOR and RVD have been reported to be dependent on cytoskeletons, here, we searched cytoskeleton-related proteins involved in regulation of VSOR and RVD in human epithelial cells. Detergent solubility assays of the microsomal fraction suggested that α-actinin 4 (ACTN4) becomes associated with cytoskeletons when Intestine 407 and HEK293T cells were exposed to hypotonic solution. Cell volume measurements demonstrated that RVD was facilitated by ACTN4 overexpression whereas RVD was suppressed by siRNA-mediated ACTN4 downregulation. Protein overlay assays revealed that ABCF2, a member of ABC transporter superfamily, is a binding partner of ACTN4. Their interaction requires the NH₂-terminal regions of both ACTN4 and ABCF2 and is enhanced by hypotonic stimulation. ABCF2 overexpression inhibited RVD, whereas its downregulation facilitated RVD after osmotic swelling. Moreover, the whole-cell VSOR current was suppressed by overexpression with ABCF2 in HEK293T cells. Thus, it is concluded that ABCF2 is an ACTN4-binding protein which is involved in regulation of VSOR and RVD. [J Physiol Sci. 2007;57 Suppl:S124]

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Rho-Kinase (ROK)-mediated Ca²⁺-independent contraction of vascular smooth muscle (VSM) plays a pivotal role in the pathophysiology of vasospasm. We previously identified sphingosylphosphorylcholine (SPC) and Fyn as upstream signaling molecules of ROK-mediated Ca²⁺-independent abnormal contraction of VSM. In addition, we found that eicosapentaenoic acid (EPA) can selectively inhibit the SPC-induced Ca²⁺-independent contraction without affecting the [Ca²⁺]_i elevation and contraction induced by high K⁺-depolarization. Moreover, we reported that EPA was clinically and highly effective in preventing vasospasm after subarachnoid hemorrhage. However, EPA is limited to oral administration and thus unsuitable for clinically serious patients unable to ingest orally. We therefore screened for novel compounds which could inhibit Ca²⁺-independent abnormal VSM contraction induced by SPC, and substitute for EPA. Tension study of VSM showed that several compounds inhibited the SPC-induced abnormal VSM contraction, to an extent comparable to the effects of EPA. These results suggest that the newly found compounds would be the candidates for novel therapeutic drugs for vasospasm and substitute for EPA. [J Physiol Sci. 2007;57 Suppl:S124]

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Using RNA interference method, we previously showed that Fyn, a member of Src family tyrosine kinase, was involved in the sphingosylphosphorylcholine (SPC)-induced formation of stress fibers. The siRNA-mediated down-regulation of Fyn tyrosine kinase partially inhibited the SPC-induced stress fiber formation. In further study, here we investigated the effects of over-expression of Fyn constructs on the assembly of actin stress fibers. The cDNAs encoding constitutively active form (ca-Fyn; FynY530F), dominant negative form (dn-Fyn; FynK298M), and wild type of Fyn (FynWT) were cloned into pcDNA6/myc-His A vector. The constructs were verified by DNA sequencing. Using Nucleofector, NIH3T3 fibroblasts were transfected with myc-tagged Fyn constructs, and their over-expressions were confirmed in western blotting using the antibody against c-myc (9E10). SPC actions were mimicked by ca-Fyn (FynY530F) and were blocked by dn-Fyn (FynK298M). Immunofluoresence analysis showed that stress fiber formation was induced by ca-Fyn (FynY530F), but was blocked by dn-Fyn (FynK298M). Stimulation of NIH3T3 fibroblasts with SPC induced stress fiber formation in the cells transfected with empty vector or FynWT, but not in the FynK298M-overexpressed cells. These findings suggest that Fyn tyrosine kinase plays an important role in actin stress fiber formation induced by SPC. [J Physiol Sci. 2007;57 Suppl:S124]

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Whereas the Ca²⁺-dependent contraction of vascular smooth muscle (VSM) regulates physiological vascular tone, the Rho-kinase (ROK)-mediated Ca²⁺-sensitization of VSM contraction contributes to abnormal VSM contraction such as vasospasm. We previously found that sphingosylphosphorylcholine (SPC) is an upstream messenger for the ROK-mediated Ca²⁺-sensitization and those inhibitors of Src family tyrosine kinase (Src-TKs) blocked the SPC-induced contraction and activation of ROK. Among Src-TKs, Fyn and c-Src were expressed in VSM, and SPC translocated Fyn, but not c-Src, from the cytosol to the cell membrane in cultured human coronary artery smooth muscle cells (CASMCs). In the present study, we attempted to determine, which molecule in CASMCs contributes to the Ca²⁺-sensitization mediated by a SPC/ROK pathway. The siRNA-mediated knockdown of Fyn inhibited the SPC-induced contraction of CASMCs. In addition, Fyn constructs [wild type, and constitutively active form (ca-Fyn), and dominant negative form (dn-Fyn)] were transfected to CASMCs with high efficiency (> 50%), although CASMCs were well-differentiated contractile cells. Overexpression of ca-Fyn induced remarkable CASMCs contraction. But, dn-Fyn did not induce any contraction. We have also attempted to design an in vitro assay to visualize the downstream molecules of the signal pathway, which will be discussed. [J Physiol Sci. 2007;57 Suppl:S124]

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Recently we have reported that sphingosylphosphorylcholine (SPC), which is a sphingolipid mediator and generated by N-deacylation of sphingomyelin, causes Rho-kinase-mediated Ca²⁺-sensitization of vascular smooth muscle, thereby leading to abnormal vascular contraction, such as cerebral vasospasm after subarachnoid hemorrhage (SAH). Indeed, SPC concentration in cerebrospinal fluid is much higher in post-SAH patients than in non-SAH patients. In addition, various vascular diseases, such as acute cerebral and myocardial infarction, are also likely to involve the vasospastic activity. Therefore, in order to assess the relationship between SPC concentration and vascular diseases, we tried to develop a rapid and accurate method to measure the serum concentration, using a reversed phase high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS). Using deuterium-labeled SPC as an internal standard, the ratio of ion abundance was determined by multiple reaction monitoring mode. The optimized HPLC/ESI-MS/MS condition was sufficient for the construction of linear calibration curve and continuous analysis. Furthermore, we examined the influence of differences in the technical procedures for the blood collection and the sample preparation and measurement on the yield and stability of SPC. [J Physiol Sci. 2007;57 Suppl:S125]

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The effect of extracellular ATP on adipogenesis was investigated using 3T3-L1 cell line. In this cell line, growth arrest is required before initiation of differentiation, and growth-arrested post-confluent cells can be converted to adipocytes by the presence of the adipogenic hormones. On the other hand, proliferating cells preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of 10-100 μM ATP underwent adipocyte differentiation mediated through P2Y receptors, suggesting that extracellular ATP renders preadipocytes responsive to the adipogenic hormones during the growing phase. To determine intracellular proteins playing a role in P2Y receptor-mediated regulation of adipocyte differentiation, we investigated ATP-induced changes in expression profiles of phosphoproteins in 3T3-L1 preadipocytes and found some phosphoproteins modulated by purinergic stimulation. One of the identified phosphoproteins using LC/MS/MS was nucleophosmin. It has been reported that nucleophosmin regulates cell cycle progression and the amount of the phosphorylated form is reduced in growth-arrested state. When the cells were incubated with 100 μM ATP, the expression of phosphorylated nucleophosmin was decreased within 5 min and recovered to the unstimulated level by 30 min incubation. The results suggest the possibility that the modification of nucleophosmin by extracellular ATP induces growth arrest of preadipocytes leading to the proceeding of the differentiation. [J Physiol Sci. 2007;57 Suppl:S125]

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Ghrelin, isolated from the human and rat stomach, is the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). We have reported that ghrelin and GHS-R were expressed in pancreatic islets and that ghrelin suppressed glucose-induced insulin release via activation of voltage-dependent delayed rectifier K⁺ channels and attenuation of glucose-induced action potentials, which leads to suppression of glucose-induced Ca²⁺ signaling in β-cells. In this study, we employed ghrelin knockout (Ghr-KO) mice and explored physiological role of ghrelin in the regulation of insulin release. Glucose-induced, but not KCl-induced, insulin release from isolated islets of Ghr-KO mice was significantly greater than that of wild-type control mice, while insulin content was unaltered. Ghr-KO mice displayed increased insulin and decreased glucose responses in glucose tolerance tests. In contrast, no significant differences in insulin tolerance tests were observed between Ghr-KO and wild-type mice. These findings reveal that the islet-originated ghrelin serves as a physiological downregulator of glucose-induced insulin release. [J Physiol Sci. 2007;57 Suppl:S125]

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The maxi-anion channels express in a large variety of cell types. Its main physiological function is considered as a pathway for the regulated release of ATP and glutamate. The mechanisms by which the maxi-anion channel is regulated remain poorly understood. In the present study, we analyzed regulation mechanisms of the maxi-anion channel in inside-out patches excised from mammary gland C127 cells exposed to artificially designed intracellular solutions. An increase in the free Mg²⁺ concentration led to a dramatic increase in the rate of channel activation with half-maximal activation at 3 mM. Mg-ATP added to the intracellular solution greatly suppressed the channel activation with half-maximal inhibition at 36 μM. A non-hydrolysable analogue of ATP, AMP-PNP, did not suppress the channel activation. Broad-spectrum tyrosine phosphatase inhibitors, such as vanadate, dephostatine, molybdate and brometetramisole, effectively suppressed the channel activation. Tyrosine kinase inhibitors, such as AG18 and genestein, but not a broad spectrum serine-threonine kinase inhibitor, H7, restored the channel activity from its inactivated state. Taken together, it is concluded that the maxi-anion channel is regulated by magnesium and ATP, and suggested that the tyrosine dephosphorylation and phosphorylation processes are involved in the channel activation and inactivation, respectively. [J Physiol Sci. 2007;57 Suppl:S125]

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Application of bradykinin (BK) induced cytosolic Ca²⁺ rise in cultured mouse cortex astrocytes as well as neurons when co-cultured with astrocytes. However, BK failed to induce any Ca²⁺ response in the neurons cultured alone. In the co-culture system, a blocker of volume-sensitive outwardly rectifying (VSOR) anion channel, phloretin or DIDS, or that of NMDA receptor, AP-5, significantly suppressed BK-induced Ca²⁺ response in neurons but not in astrocytes. Also, astrocytes, but not neurons, were found to respond to BK with activation of whole-cell chloride currents. Although BK-stimulated astrocytes did not exhibit significant cell swelling, BK-activated anionic currents showed the properties phenotypic of VSOR anion channel; that is, moderate outward rectification, inhibition by osmotic shrinkage, sensitivity to phloretin and DIDS, dependence of intracellular ATP, and permeability to glutamate. BK stimulation was also found to induce release of glutamate, but not ATP, from astrocytes in a manner sensitive to phloretin, hypertonicity and blockers of BK receptors. These results suggest that glutamate released from astrocytes via their VSOR anion channels serves as a signal mediating the BK-induced glia-neuron interaction. [J Physiol Sci. 2007;57 Suppl:S126]

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Barrier function of the skin is essential to protect against infection and dehydration. In addition to a hydrophobic cornified cell layer, formation of adherence- and tight-junctions among the keratinocytes also form a significant barrier. Differentiation and formation of these junctions need an increase in intracellular Ca²⁺, however, the molecular mechanism remains poorly understood. TRPV4 is a Ca²⁺-permeable ion channel, which is known to be expressed in keratinocytes, and activated by warm temperatures (–33°C). Therefore, we examined the role of TRPV4 in the differentiation process of keratinocytes and epidermis using TRPV4-KO mice. Yeast two-hybrid screening identified an interaction between TRPV4 and β-catenin, both of which were observed at cell-cell junction areas in keratinocytes. Reorganization of actin cytoskeleton in TRPV4-KO keratinocytes was delayed and immature. An in vitro barrier function assay of a keratinocyte sheet revealed higher permeability of dextran across the sheet in TRPV4-KO keratinocytes. Barrier function in vivo was also impaired. Change in basal Ca²⁺ level after addition of extracellular high Ca²⁺ under 33°C was significantly lower in TRPV4-KO keratinocytes. Therefore, we hypothesize that TRPV4 is an important player for Ca²⁺ entry in keratinocytes that is necessary for differentiation and formation of cell-cell junctions. [J Physiol Sci. 2007;57 Suppl:S126]

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An increase of extracellular ATP concentration is observed during brain ischemia, but the origin of released ATP is not well understood. We investigated the mechanisms of ATP release from rat hippocampal slice cultures under ischemic conditions. When slice cultures were subjected to oxygen-glucose deprivation (OGD), significant release of ATP was observed within 10 min. OGD-induced ATP release was almost completely prevented by pretreatment with tetanus toxin (TeTX) and was largely suppressed by the removal of extracellular Ca²⁺ or by the application of TTX or an NMDA receptor antagonist, MK-801, whereas it was slightly diminished by Gd³⁺, a blocker of maxi-anion channels which are known to mediate glial ATP release. Slice cultures also responded to glutamate exposure for over 10 min with gradual release of ATP. Glutamate-induced ATP release was partially sensitive to TeTX and was markedly suppressed by combined application of MK-801 and a non-NMDA receptor antagonist, CNQX. Neuron-depleted slice cultures responded to glutamate exposure, but not to OGD, with ATP release. Glutamate-induced ATP release from neuron-depleted slices was little sensitive to TeTX but was significantly suppressed by Gd³⁺. Taken together, it is suggested that ischemic insult-induced ATP release from slice cultures comprises two components: one is the rapid release caused by exocytosis of neuronal synaptic vesicles containing ATP and glutamate, and another is the glutamate-dependent gradual release via Gd³⁺-sensitive glial pathways, presumably maxi-anion channels. [J Physiol Sci. 2007;57 Suppl:S126]

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It is known that human epithelial HeLa cells exhibit cell volume regulation after osmotic shrinkage, called the regulatory volume increase (RVI), by both mechanisms mediated by activation of hypertonicity-induced cation channels (HICC) and by parallel activation of Na⁺/H⁺ exchangers and anion exchangers. To analyse the signalling network behind the HICC activation, in the present study, we examined the effects of a variety of high-specificity blockers of signalling molecules on activation of HICC monitored by whole-cell patch-clamp recordings and on the RVI monitored by electronic cell sizing. These data suggest that HICC currents and HICC-mediated RVI involve tyrosine kinases, G-proteins, PLC, PKC and p38MAP kinase, and also they appear to depend on an intact actin cytoskeleton. In contrast, RhoA, PI 3-kinase, ERK 1/2, JNK 1/2 as well as exocytotic insertion of channels into the plasma membrane do not appear to participate in the signalling machinery. [J Physiol Sci. 2007;57 Suppl:S126]

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Changes in G-proteins associate with some diseases. Here we examined whether expression of Gq/11 in the brain, lung, heart, liver, kidney and small intestine alter during acute lung injury (ALI). The concentrations of Gq/11 in six organs were measured by Western blot. ALI model in rats was established by intravenous injection of oleic acid (OA, 0.2ml/kg) through tail vein. All rats were randomly divided into a vehicle injected control group (C group) and experimental groups (OA groups). OA groups included 4 sub-groups: OA30min, OA60min, OA90min, and OA120min. Blood gas indexes (pH, PaO₂, PaCO₂), cardiac function indexes (MAP, LVSP, ±LVdp/dtmax), hepatic function indexes (ALT, AST), renal function indexes (BUN, Cr) and enzyme indexes (MDA, LDH, CK) of plasma and organs were measured. Each group was examined by microscopy. Compared with C group, expression of Gq/11 in OA groups increased by 20-70% in all organs with time passing. PaO₂, MAP, LVSP and ±LVdp/dtmax gradually decreased, BUN and Cr in the plasma notably increased. ALT, AST, MDA contents and LDH activity increased with time passing. CK activity in the plasma, brain and heart of OA groups notably increased. Histological changes were found by microscopy. We conclude that up regulation of Gq/11 protein concentration in the organs may play a role in damaged multiple organs during ALI. Change of signal transduction mediated by Gq/11 protein may take part in the development of ALI. [J Physiol Sci. 2007;57 Suppl:S127]

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Immunosuppressant such as cyclosporine A and tacrolimus is used to suppress the immune response for the therapeutic purposes of organ and tissue transplantation. However, clinical application of these drugs is limited by severe side effects. One of harmful effect is hypomagnesemia which may be caused by a reduction of renal magnesium reabsorption. In the present study, we examined the effect of cyclosporine A on the expression of magnesium channel TRPM6 and TRPM7 in renal epithelial NRK-52 cells. In RT-PCR and Western blotting analysis, cyclosporine A dose-dependently decreased in TRPM6 without TRPM7. The addition of extracellular MgCl₂ under the Mg²⁺-free conditions caused an increase of intracellular free Mg²⁺ concentration ([Mg²⁺]i). The increase of [Mg²⁺]i was significantly inhibited by cyclosporine A. Gene silencing using siRNA against TRPM6 caused a decrease of TRPM6 without TRPM7. The increase in [Mg²⁺]i caused by MgCl₂ addition was inhibited by siRNA transfection. Next, we investigated the effect of cyclosporine A on the expression of transcriptional factors, c-fos and c-jun. Cyclosporine A dose-dependently decreased in c-fos without c-jun. We suggest that cyclosporine A causes the reduction of renal magnesium reabsorption mediated by decrease in TRPM6 and c-fos expressions, resulting in a hypomagnesemia. [J Physiol Sci. 2007;57 Suppl:S127]

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The purpose of this study is to elucidate whether two types of phenoxazine derivatives, 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one (Phx-1) and 2-amino-phenoxazine-3-one (Phx-3) induce caspase-dependent or independent cell death of human glioblastoma cell lines, A-172 and U-251 MG. Phx-1 and Phx-3 were synthesized by the reactions of bovine hemoglobin with 2-amino-5-methylphenol and o-aminophenol, respectively. These phenoxazines extensively suppressed the proliferation of A-172 and U-251 MG cells (50% inhibition concentration (IC₅₀) of Phx-1: 60 μM in both lines and IC₅₀ of Phx-3: 10 and 3 μM in A-172 and U-251 MG cells, respectively). Phx-1 and Phx-3 increased predominantly the population of both annexin V- and propidium iodide-positive cells in both lines, indicating that these phenoxazines induce cell death at late phase apoptosis/necrosis. Phx-1 and Phx-3 markedly increased the activity of caspase-3/7 in both lines. However, a pan-caspase inhibitor, z-VAD-fmk was not able to attenuate anti-proliferative and apoptotic/necrotic activities of Phx-1 and Phx-3 against both lines, although completely inhibited the activation of caspase-3/7. These results suggest that phenoxazine derivatives, particularly Phx-3 exerts strong anti-proliferative and apoptotic/necrotic effects on human glioblastoma cell lines, A-172 and U-251 MG independent of caspase family signaling cascades. [J Physiol Sci. 2007;57 Suppl:S127]

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