Cisplatin is a potent antitumor drug but its clinical use is limited by nephrotoxic side effects. Cellular and molecular mechanisms of cisplatin-induced nephrotoxicity have not been known well. We examined whether nitric oxide (NO) is involved in the cisplatin-induced nephrotoxicity and whether sodium-dependent glucose transporter (SGLT1) has cytoprotecitve effect. In porcine renal epithelial LLC-PK1 cells, cisplatin induced necrosis at high concentration as assessed by propidium iodide stain. The proportion of necrosis in sub-confluent condition is higher than that in the confluent condition. This cell line induces expression of SGLT1 after reaching the confluent condition. Interestingly, phloridzin, a potent inhibitor of SGLT1, enhanced cisplatin-induced necrosis in the confluent condition. Next, we checked the effect of cisplatin on transepithelial electrical resistance (TER). Cisplatin decreased TER in a time-dependent manner and disrupted ZO-1 distribution at tight junction. Phloridzin enhanced the decrease of TER by cisplatin. Cisplatin increased the productions of reactive oxygen species. Among them, NO production was enhanced by phloridzin. The cisplatin-induced decrease of TER was partially blocked by NG-nitro-L-arginine, a NO synthase inhibitor. These results suggest that NO is concerned in the cisplatin-induced nephrotoxicity and SGLT1 endogenously reduces cellular injury mediated by suppression of NO production. [Jpn J Physiol 55 Suppl:S70 (2005)]

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[Objective] The glycolipid- and cholesterol-rich membrane microdomains are lateral components of the plasma membranes and considered to function as platforms for both signal transduction and membrane trafficking. However, role of membrane microdomains in salivary glands has not been elucidated. In this study, we isolated membrane microdomains of rat parotid secretory granules using sucrose density ultracentrifugation, and compared the components of membrane microdomains in the process of the granule maturation. [Methods] Secretory granules in the rat parotid gland were purified using percoll. The components of membrane microdomains are biochemically separated as detergent-resistant membranes (DRMs) based on their insolubility in the detergent. [Results] When DRMs separated using detergent 1% Triton X-100, Syntaxin 6, VAMP-2 and munc-18 were recovered in GM1a-rich DRMs fraction. On the other hand, when DRMs separated using detergent 1% Brij-58, Syntaxin 6 detected in the different fraction from GM1a-rich one. In the parotid gland of rat treated with isoproterenol for 8 hours, formation of new secretory granules was observed with electron microscopy. In the granule Brij-58 insoluble DRMs, Syntaxin 6 was distributed in GM1a-rich fraction. [Conclusion] These results suggest that Syntaxin 6 exists on GM1a-rich microdomains in immature granule. For the granule maturation, exclusion of Syntaxin 6 from GM1a-rich microdomain appears to be important. [Jpn J Physiol 55 Suppl:S71 (2005)]

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The periodontal ligament fibroblasts (PDL fibroblasts), the principal cells in PDL, are responsible for PDL functions. In this work, we examined the properties of mechanosensitive TREK-1 K⁺ channels (a member of the two-pore-domain K⁺ channel family) in cultured human PDL fibroblasts. Single-channel currents of TREK-1 channels were recorded by patch-clamp single-channel-recording techniques. The single-channel conductance of TREK-1 channel was 100 pS in symmetrical K⁺-rich solutions. The open probability of the channel was low at the quiescent state (P_o=0.012±0.004, n=44), but it was strongly increased by the application of membrane stretch. Unsaturated fatty acids, including arachidonic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, oleic acid, DHA, and EPA dose-dependently enhanced the channel activity (P_o=0.247±0.069 by 10 μM arachidonic acid, n=8), but saturated fatty acids, such as palmitic acid, stearic acid, and arachidic acid had no effects. The channel activity was also increased by acid solution (P_o=0.211±0.036 at pH 6.0, n=7), and temperature (P_o=0.072±0.043 at 50°C, n=3). The channel activity, which had been enhanced by 10 μM arachidonic acid, was decreased by the application of catalytic subunit of protein kinase A at 20 U/ml (P_o=0.028±0.018, n=3). The channel was partially blocked by quinine, quinidine, and Ba²⁺, but was not affected by tetraethylammonium, Cs⁺, and 4-aminopyridine. Immunocytochemical observation showed that TREK-1 K⁺ channel proteins were present in PDL fibroblasts. [Jpn J Physiol 55 Suppl:S71 (2005)]

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Stimulation of phospholipase C-coupled receptor induces a release of Ca²⁺ from the intracellular stores and subsequent influx of Ca²⁺ across the plasma membrane, termed store-operated Ca²⁺ entry (SOCE). SOCE is also induced by inhibition of sarcoplasmic endoplasmic reticulum Ca²⁺ pump (SERCA). One of the hypothesized mechanisms by which the state of filling of the stores is communicated to the plasma membrane is direct or indirect association of two membranes. Noradrenaline is known to induce a rapid [Ca²⁺]_i increase followed by the activation of SOCE in brown adipocytes. To investigate spatial changes in [Ca²⁺]_i during the activation of SOCE, confocal line scanning of [Ca²⁺]_i was performed in fluo-3-loaded rat brown adipocytes. Linescan images were acquired by repeatedly scanning the laser beam along with a single line at 5-200 msec intervals. When the cells were exposed to 1 μM noradrenaline, rapid [Ca²⁺]_i increase was observed in intracellular space in close vicinity to the plasma membrane followed by the increase in whole cytoplasmic space, indicating the possibility that ER locate close to the plasma membrane. On the other hand, SERCA inhibitor thapsigargin (100 nM) firstly induced a slower rise in the central area distanced from the plasma membrane followed by the gradual spreading of Ca²⁺ increase in whole cytoplasmic space. These results suggest that the activation mechanism of SOCE induced by noradrenaline is different from that induced by thapsigargin. [Jpn J Physiol 55 Suppl:S71 (2005)]

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We previously isolated a cDNA encoding a large GTP-binding protein (mOPA1) from the mouse brain, and observed that mOPA1 is localized to mitochondria and its expression induced the mitochondrial fragmentation in transfected cultured cells. By probing mouse brain extracts on SDS-PAGE with two kinds of anti-mOPA1 antibodies that recognize the C-terminal region of mOPA1, we detected two bands of approximately 90 and 80 kDa. Highly similar results were observed for COS-7 or HEK293T cells transfected with wild type mOPA1. Since their sizes were obviously smaller than the estimated molecular weight (111 kDa) of mOPA1, a possibility of intracellular processing was suggested. Judging from the band size of N-terminal deletion mutants, mOPA1 was showed to cleave at the N-terminal region near 100 or 200 residues. We aimed to determine the cleavage positions and analyzed the processing pattern of various point mutants and deletion mutants of mOPA1. Both 90 kDa and 80 kDa bands almost completely disappeared by R101S point mutation or by T191-A195 deletion, respectively. These results show that intracellular processing of the mOPA1 protein occur at two positions near R101 and T191 residues, producing 90 and 80 kDa forms. [Jpn J Physiol 55 Suppl:S71 (2005)]

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Hyperlipidemia is a major risk factor of cardiovascular events. We previously identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as upstream mediators of the Rho-kinase (ROK)-mediated Ca²⁺ sensitization of vascular smooth muscle (VSM), which plays a pivotal role in abnormal VSM contraction such as vasospasm. In the present study, we examined the possible link between hyperlipidemia (or cholesterol) and the SPC/Src-TK/ROK-mediated Ca²⁺ sensitization. In VSM strips obtained from hyperlipidemic patients and rabbits, the extent of the SPC-induced Ca²⁺ sensitization correlated well with total serum cholesterol level and was diminished by either cholesterol-lowering therapy or in vitro selective depletion of cholesterol in VSM tissue by β-cyclodextrin (β-CD). Immunofluorescent study using cultured VSM cells under confocal microscope showed that SPC induced the translocations of fyn, a member of Src-TK, and ROK from cytosol to cell membrane, where they were colocalized with markers of membrane lipid rafts (a cholesterol-enriched membrane microdomain) such as caveolin-1 and cholera toxin subunit B. β-CD treatment markedly reduced the labeling of these raft markers and diminished the SPC-induced translocations of fyn and ROK from cytosol to membrane lipid rafts. These findings suggest that cholesterol and its enriched membrane lipid rafts may contribute to the SPC/Src-TK/ROK-mediated Ca²⁺ sensitization of VSM. [Jpn J Physiol 55 Suppl:S72 (2005)]

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Stress fiber formation plays an important role in the regulation of the cell structure and motility. At present, Rho GTPase and its effecter Rho-kinase (ROK) are known to play a critical role in stress fiber formation. However, the upstream signaling pathway for the activation of Rho GTPase and ROK remains to be elucidated. We previously showed that sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) are the upstream mediators of ROK-mediated Ca²⁺ sensitization of vascular smooth muscle contraction. Here we found that SPC induced stress fiber formation in NIH3T3 fibroblasts, which was assessed by the confocal images of the F-actin staining with phalloidin. The SPC-induced stress fiber formation was abolished by a ROK inhibitor (Y27632) and was partially blocked by Src-TK inhibitors (PP1, PP2), but not by their inactive analogue (PP3). In contrast, the stress fiber formation induced by its well-known stimulant, lysophosphatidic acid (LPA) was blocked by Y27632, but not by PP1 or PP2, although SPC and LPA induced stress fiber formation to the same extent. In addition, SPC, but not LPA, induced the formation of filopodia-like protrusions, which were not blocked by PP1, PP2, and Y27632. These findings suggest that SPC and LPA stimulate the ROK-mediated stress fiber formation in fibroblasts through the Src-TK-dependent and independent pathway, respectively. The formation of filopodia-like protrusions induced by SPC, but not by LPA, was not mediated by the both kinases and its mechanism is unknown. [Jpn J Physiol 55 Suppl:S72 (2005)]

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Interstitial application of Mn²⁺ ion can be used to visualize neural activities in the brain by using T₁-weighted MRI. When we infuse Mn²⁺ in the cerebrospinal fluid (CSF), Mn²⁺ can be transferred into the interstitial space of brain through the ependymal cells. In order to optimize this entry process, we have analysed diffusion process of Mn²⁺ into the parenchyma. Mn²⁺ solution infused from the lateral ventricle of the rat brain, diffusion of Mn²⁺ was measured perpendicular direction from the third ventricle. T₁ relaxation times were measured at 4.7 T animal MR imaging system. Relaxivity of Mn²⁺ was used to convert T₁ relaxation time to Mn²⁺ concentration. From these results and the simple diffusion equation, apparent diffusion coefficient (Dap) of Mn²⁺ ion in the interstitial space is 2.5 x 10⁻³ mm²/min. However, the Dap tends to decrease when the distance from the third ventricle increased. It might be possible that 1) Mn²⁺ ion is trapped by neural/glial cells during diffusion, and/or 2) a presence of axial flux of the interstitial fluid in the brain. [Jpn J Physiol 55 Suppl:S72 (2005)]

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Exocrine glands keep the cell polarity that is due to the tight junctions that partition a cell surface into apical and basolateral membranes. In the parotid gland, the transmembrane protein occludin is observed to form tight junctions along the intercellular canaliculi. Actin filaments run along the tight junction and aquaporin5 is specifically detected at the apical membrane. The polarity is important for oriented water and electrolyte transport. We have established a method to culture dispersed parotid acinar cells that retain their ability to generate new secretory granules and secrete amylase upon the stimulation. In this study, we analysed the cell polarity of the primary culture. After the culture for 24 h, since aquaporin5 was seen to have diffused to the plasma membrane, the apical membranes could not be distinguished. No occludin was detected, suggesting that the tight junction was broken in the primary culture. These results suggest that the polarity of the cells was lost. In contrast, at 48 h after the dispersion, the cells spread as a monolayer and occludin bands appeared again between the cells. Immunoblotting with anti-occludin antibody showed that the expression of occludin increased time-dependently although it was once lost after the dispersion. These results suggest that a kind of polarity may have begun to return. [Jpn J Physiol 55 Suppl:S72 (2005)]

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A Rho-kinase (ROK)-mediated Ca²⁺ sensitization of vascular smooth muscle (VSM) contraction plays a critical role in vasospasm. We previously identified sphingosylphosphorylcholine (SPC) as the upstream mediator for the ROK-mediated Ca²⁺ sensitization of VSM contraction. Although the membrane receptors for SPC were reported as the [Ca²⁺]i-elevating G-protein-coupled receptors (GPCRs), SPC-induced contractions of intact VSM was not accompanied with any elevation of [Ca²⁺]i and SPC induced contraction of membrane-permeabilized VSM in the absence of cytosolic GTP which is required for the activation of G-proteins and thus of GPCRs. These findings are compatible with the interaction of SPC with the other membrane components than GPCRs or the direct interaction between SPC and lipid membrane, which may in turn affects the functions of membrane signaling molecules. In addition, we observed the strong link between the SPC-induced contraction and the tissue and cellular cholesterol in VSM, suggesting the possible roles of cholesterol-enriched membrane microdomains, membrane lipid rafts, in the SPC/ROK-induced Ca²⁺-sensitization of VSM contraction. Thus, we investigated the direct interaction of SPC with model membranes. The measurements of surface plasmon resonance using BIACORE system revealed that SPC highly associates with the model membrane microdomains, lipid rafts and that cholesterol in the model membrane enhances the incorporation of SPC into the membrane. [Jpn J Physiol 55 Suppl:S73 (2005)]

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The Cell shrinkage is known to decrease intracellular Cl⁻ concentration [Cl⁻] _i. Cell shrinkage enhances Ca²⁺ regulated exocytosis in gunia pig antral mucous cells. In the present study, the effects of [Cl⁻]_i on the Ca²⁺ regulated exocytosis were examined, by replacing Cl⁻ of the perfusion solution with NO₃. The exocytotic events in gunia pig antral mucous cells were observed using video-microscopy. The Ca²⁺-regulated exocytosis was activated by acetylcholine (ACh). The Ca²⁺-regulated exocytosis consisted of two phases; an initial transient phase followed by a sustained phase. The replacement of Cl⁻ with NO₃ caused the both phase to increase, and shifted the ACh dose response curve to the lower concentration side. Similar enhancement was induced by bumetanide (an inhibitor Na⁺/K⁺/Cl⁻ cotransport, 20 μM). Thus, inhibition of Cl⁻ influx appears to enhance the ACh-evoked exocytotic events. Contrary NPPB (Cl⁻ channel blocker 2 μM), which inhibits Cl⁻ efflux, decreased the frequency of ACh-stimulated exocytosis and suppressed the enhancement induced by the Cl⁻-free solution. These results indicate that inihibition of Cl⁻ influx increases Ca²⁺ regulated exocytosis, contrary, inhibition of Cl⁻ efflux decreases it. Based on the observation, [Cl⁻]_i appears to play an important role in activation of Ca²⁺ regulated exocytosis. [Jpn J Physiol 55 Suppl:S73 (2005)]

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Phospholipase C-zeta (PLCζ), a novel isoform of PLC, receives much attention, because it is a strong candidate of the mammalian sperm factor that is driven into the egg cytoplasm upon sperm-egg fusion at fertilization and induces Ca²⁺ oscillations leading to egg activation. We have synthesized recombinant PLCζ using baculovirus/Sf9-cell expression system and showed that PLCζ can induce Ca²⁺ oscillations at low concentrations probably via IP₃-induced Ca²⁺ release when injected into mouse eggs. We also showed that PLCζ has such a high Ca²⁺-sensitivity of PLC activity that the enzyme can be active in resting cells at ∼100 nM Ca²⁺. The structure-function relationship was examined by mutational analysis. Deletion of EF1 and EF2 of N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate-hydrolysing activity in vitro and loss of Ca²⁺ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 little affected the Ca²⁺-sensitivity of PLC activity, while deletion of EF1 to EF3 caused 12-fold elevation of EC50 of Ca²⁺ concentration. Thus, EF1 and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca²⁺ sensitivity. Deletion of four EF-hand domains or C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisite for the PLCζ activity. [Jpn J Physiol 55 Suppl:S73 (2005)]

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Mucin exocytosis from antral mucous cells of guinea pig is maintained by an autocrine mechanism via prostaglandin E₂ (PGE₂) during acetylcholine (ACh) stimulation. Indomethacin (IDM) or aspirin (ASA) partially inhibited the frequency of ACh -stimulated exocytotic events. However, the IDM-induced decrease in the frequency of ACh-stimulated exocytotic events was 40%, while ASA-induced decrease was 60%. Inhibition of PKA effects by ONO8713 (100 μM, an inhibitor of EP receptors) or H-89 also decreased the frequency by 60%. The COX synthesizes PGE₂ and 15(R)-HPETE from arachidonic acid (AA), and IDM inhibits both productions, however, ASA inhibits only PGE₂ production. This suggests that IDM may accumulate AA. In ASA-treated cells, IDM increased the frequency of ACh-stimulated exocytotic events similar to that observed in IDM-treated cells. IDM alone increased the frequency of exocytotic events. The IDM-induced increases, which were eliminated by AACOCF3 (100 μM, an inhibitor of phospholipase A₂), were mimicked by AA. Moreover, AA effects were mimicked by WY14643 (an agonist of peroxisome proliferator-activated receptors, PPARs). The present study suggests that accumulation of AA may enhance the Ca²⁺-regulated exocytosis via PPARs in IDM-treated antral mucous cell. [Jpn J Physiol 55 Suppl:S73 (2005)]

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We have recently demonstrated in VSM that membrane depolarization (KCl stimulation) induces Ca²⁺-dependent Rho activation and MP inhibition through the mechanisms involving Rho kinase (ROK). ROK is shown to mediate MP inhibition by phosphorylating 110-kDa myosin targeting regulatory subunit MYPT1 of MP and/or MP-inhibitory phosphoprotein CPI-17 in receptor agonist-stimulated VSM. Protein kinase C (PKC) might also phosphorylate CPI-17, thus contributing to MP inhibition. However, it is unknown whether and how cyclic AMP, which induces inhibition of contraction, affects Ca²⁺-dependent Rho activation and MP inhibition. KCl induced phosphorylation of both MYPT1 and CPI-17 in a ROK inhibitor-sensitive manner, suggesting that Rho kinase mediated phosphorylation of CPI-17 as well as MYPT1. Forskolin, an adenylate cyclase activator, inhibited KCl-induced contraction with inhibition of MLC phosphorylation. Forskolin also suppressed KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. KCl-induced Rho activation and contraction was inhibited by phosphoinositide 3-kinase (PI3K) inhibitors. KCl induced stimulation of the activity of a PI3K isoform. We are now investigating the effect of forskolin on KCl-induced PI3K activation. Thus, membrane depolarization induces not only Ca²⁺-dependent MLC kinase activation, but also Ca²⁺-dependent MP inhibition through a PI3K, Rho and Rho kinase. Cyclic AMP effectively inhibits Rho activation and thereby MP inhibition. [Jpn J Physiol 55 Suppl:S74 (2005)]

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Phospholipase C-zeta (PLCζ) is a strong candidate of the mammalian sperm factor that causes IP₃ receptor-mediated repetitive Ca²⁺ release in the egg at fertilization and triggers egg activation. Evidence suggests that the sperm factor is driven into the egg cytoplasm upon sperm-egg fusion and then into the male and female pronuclei (PN) formed after egg activation. Ca ²⁺ oscillations cease upon PN formation 5-6 h after fertilization, possibly because the sperm factor in the egg cytoplasm is removed by sequestration into the PN. The nuclear translocation of PLCζ was continuously observed, when RNA encoding PLCζ fused with a fluorescent protein 'Venus' was injected into unfertilized mouse eggs and induced Ca²⁺ oscillations and egg activation. PLCζ accumulated in the (female) PN was seen between 5 and 15 h after RNA injection, and was lost upon breakdown of the nuclear envelop at ∼16 h just before cell division. After the first cleavage, PLCζ was accumulated again into the nuclei of two blastomeres. This was observed as well in the 4-cell, 8-cell, 16-cell embryo and the morula after fertilization, when PLCζ-Venus RNA was injected into a blastomere of the 2-cell or 4-cell embryo in which the nucleus was clearly seen in the bright field. Thus, the ability of nuclear accumulation of PLCζ is preserved during early embryonic development. The nuclear translocation of PLCζ might regulate cell cycle-dependent Ca²⁺ oscillations in mouse embryos. [Jpn J Physiol 55 Suppl:S74 (2005)]

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[Purpose] The stereoisomers of D-glucoses are expected to have many physiological activities because of the similarities of the structures, though most of these are rarely found in the nature. Angiogenesis plays an important role in tumor growth and metastasis. To estimate the applicability of monosaccharides to anti-cancer drugs, we examined the effects of monosaccharides on the proliferation, the tube formation and the glucose uptake of human umbilical vein endothelial cells (HUVEC). Thirty six kinds of monosaccharides were used and the data were correlated with their structures. [Methods and Results] The proliferation assay was performed 48 h after the addition of sugars (0–20 mM) to the culture medium. Seven sugars were found to inhibit the proliferation in a dose dependent manner. The tube formation assay was performed by CD31 staining of endothelial cells 10 days after the addition of sugars (0–20 mM) to the co-culture system of HUVEC and human fibroblast. Fourteen sugars inhibited the tube formation in a dose dependent manner. To estimate the glucose uptake, ¹⁴C labeled D-glucose was added to the culture medium with and without other sugars, and the uptake was calculated at 48 h. Three sugars inhibited the glucose uptake in HUVEC. [Conclusion] Comparing the stable structure of the sugars, following common structures seem to be required to inhibit the proliferation or tube formation of HUVEC: (i) Less 1,3-diaxial interaction in the pyranose form; and (ii) Equatorial substituent at C₂ or C₃ is H (hydrogen). [Jpn J Physiol 55 Suppl:S74 (2005)]

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Chromaffin cells in the perfused rat adrenal medulla were co-loaded with Fura-2 and Rhod-2 for respectively monitoring changes in the free Ca²⁺ concentration in the cytosol, [Ca²⁺]_c and that in mitochondria, [Ca²⁺]_m. When cells were stimulated for 10s with a perfusate containing 40 mM K⁺, [Ca²⁺]_m increased to a maximum level at the end of the stimulation period and then decreased to the resting level with a time course much slower than that of the simultaneously observed [Ca²⁺]_c response. With increasing the stimulation period up to 30s, the duration of [Ca²⁺]_m response increased markedly without changing its maximum level. To perform computer simulation to reproduce such characteristics of [Ca²⁺]_m responses, mitochondrial functions including Ca²⁺ uptake by uniporter, buffering of Ca²⁺ in the matrix and Ca²⁺ extrusion by Na⁺/Ca²⁺ exchanger were additionally implemented in a previously constructed model of a chromaffin cell. In simulation, a model involving precipitation of calcium phosphate salt in the mitochondrial matrix, but not with high concentrations of a soluble Ca²⁺ buffer with various affinities, allowed to reproduce the observed characteristics of [Ca²⁺]_m responses. Rotenone, which depolarizes the mitochondrial membrane potential, was used in expectation that it may reduce the Ca²⁺ clearance by mitochondria to increase secretory responses. Such expectation was in fact demonstrated in both experiments and simulations, though the extent of increase in secretory responses by rotenone was shown to be much lesser in experiments. [Jpn J Physiol 55 Suppl:S74 (2005)]

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InsP₃Rs were reported to be present not only in the ER, but also in secretory granules in bovine adrenal chromaffin cells. Thus, we investigated immunocytochemically which organelles contained InsP₃Rs in rat adrenal chromaffin cells. The distribution of BODIPY-InsP₃, which is a InsP₃ conjugated with a fluorescent molecule, was consistent with that of the ER identified by using an anti-calnexin Ab. Anti-InsP₃R type 2 Abs of Sigma and Chemicon each produced immunoreactive materials in agreement with BODIPY-InsP₃ binding sites. On the other hand, part of materials immunoreactive to the anti-InsP₃R2 mAb (KM1083) at the cell periphery were stained by using an anti-dopamine-β-hydroxylase Ab, but not by BODIPY-InsP₃. BODIPY-thapsigargin binding sites were consistent with distribution of the ER identified by the anti-calnexin Ab, and a prior application of thapsigargin significantly eliminated BODIPY-thapsigargin bindings. Application of muscarine failed to induce a Ca²⁺ mobilization in the presence of thapsigargin, suggesting that muscarine mobilized Ca²⁺ ions from thapsigarin-sensitive sites. The anti-InsP₃R2 Ab of Chemicon recognized a protein with about 250 kDa alone, whereas KM1083 detected an about 200 kDa protein as well as the 250 kDa in a crude membrane fraction of the adrenal medulla. The results indicate that the InsP₃R2 is present in the ER, but not in secretory granules in rat adrenal chromaffin cells. [Jpn J Physiol 55 Suppl:S75 (2005)]

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Recently we have demonstrated that in HeLa cells staurosporine (STS) rapidly exhibits the apoptotic volume decrease by activation of volume-sensitive outwardly rectifying Cl⁻ channels in a manner dependent on reactive oxygen species (ROS) and that STS-induced apoptosis of the cells is rescued by a Cl⁻ channel blocker or a ROS scavenger (PNAS 101, 6770, 2004). In the present study, thus, we examined whether apoptosis of cardiomyocytes induced by ischemia/reperfusion (I/R) is also dependent on the Cl⁻ channel and ROS. Neonatal mouse cardiomyocytes in primary culture were subjected to the I/R stress by incubating for 6 h under the hypoxic and glucose-free conditions followed by culturing for 96 h in the presence of oxygen and glucose. By this I/R stress, reduction of cell viability, DNA laddering and caspase-3 activation were induced. When a Cl⁻ channel blocker (DIDS or NPPB) was applied for the R period (but not the I period), cardiomyocyte apoptosis was largely prevented. A ROS scavenger (Tiron, N-acetylcysteine or catalase) was also effective in rescuing the cells from apoptosis when applied during reperfusion. In addition, a large amount of ROS was found to be produced upon reperfusion. These results suggest that I/R-induced cardiomyocyte apoptosis involves the Cl⁻ channel activity and the action of ROS produced upon reperfusion. [Jpn J Physiol 55 Suppl:S75 (2005)]

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A stilbene derivative, DIDS, suppresses staurosporine (STS)-induced apoptosis in many cell types. However, it has not been clarified which is the target of DIDS in apoptosis of cardiomyocytes, Cl⁻/HCO₃⁻ exchangers or Cl⁻ channels. To answer this question, we examined STS-induced reduction of cell viability, DNA laddering and caspase-3 activation using cultured mouse ventricular myocytes in the presence or absence of HCO₃⁻. STS-induced apoptosis and its DIDS sensitivity in HCO₃⁻-free conditions were indistinguishable from those in HCO₃⁻-rich conditions. Apoptosis was also prevented by application of another Cl⁻ channel blocker, NPPB, which cannot block Cl⁻/HCO₃⁻ exchangers. These data rule out contribution of Cl⁻/HCO₃⁻ exchanger in cardiomyocyte apoptosis and its DIDS sensitivity. Cardiomyocytes derived from ClC-3-deficient mice similarly underwent apoptosis after exposure to STS. Application of DIDS or NPPB prevented STS-induced apoptosis in ClC-3-deficient cardiomyocytes. Thus, we conclude that apoptosis in cardiomyocytes is critically dependent on the activity of Cl⁻ channel which is distinct from ClC-3, but not of Cl⁻/HCO₃⁻ exchangers. [Jpn J Physiol 55 Suppl:S75 (2005)]

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Cardiomyocytes are known to swell in various pathophysiological conditions such as congestive cardiac failure, ischemia, endotoxic shock and dilated or hypertrophic cardiomyopathies. In the present study, we made 3-dimensional imaging of the substrate-attached neonatal rat cardiomyocytes in primary culture using the Scanning Ion Conductance Microscopy (SICM) technique in which a fine-tipped patch pipette is employed as a probe to monitor the cell surface. Hypotonic stimulation caused cell swelling followed by a slow recovery of cell volume (regulatory volume decrease: RVD), which was impaired in the presence of NPPB and phloretin suggesting an involvement of volume-sensitive outwardly rectifying (VSOR) chloride channel therein. Conventional whole-cell recordings revealed large tamoxifen- and phloretin-inhibitable VSOR chloride currents. We next used the same pipette for both imaging the cell by SICM and for patching the specified regions on the cell by a "smart-patch" technique. These on-cell experiments revealed swelling-induced activation of single VSOR channels which exhibit outward rectification, inactivation at large positive potentials and sensitivity to phloretin and tamoxifen. The single VSOR channel activity was found to be evenly distributed over the cell surface membrane of neonatal cardiomyocytes. [Jpn J Physiol 55 Suppl:S75 (2005)]

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Intracellular Mg ²⁺ concentration ([Mg²⁺]_i) was measured with the fluorescent indicator furaptra (mag-fura-2) at 25°C. After the cells were loaded with Mg²⁺ in a high-Mg²⁺ and low-Na⁺ solution for 3 h, reduction of extracellular Mg²⁺ to 1 mM caused a rapid decrease in [Mg²⁺]_i in the presence of 140 mM Na⁺ and 0.1 mM EGTA (extracellular Na⁺-dependent Mg²⁺ efflux). To study the effects of intracellular Ca²⁺ concentration on the Mg²⁺ efflux, the initial rate of decrease in [Mg²⁺]_i (initial Δ[Mg²⁺]_i/Δt) was measured in the cells heavily loaded with a Ca²⁺ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxy-methyl ester for 3.5 h. Intracellular loading of the Ca²⁺ chelator did not significantly change the initial Δ[Mg²⁺]_i/Δt: 109.7±8.2% (7 BAPTA-loaded cells) and 102.2±10.3% (4 dimeth yl BAPTA-loaded cells) of the control measured in the absence of the intrace llular chelator. When the Mg²⁺ efflux was induced at various extracellular Ca²⁺ concentrations ([Ca²⁺]_o) and in the absence of the intracellular Ca²⁺ chelator, the initial Δ[Mg²⁺]_i/Δt values were essentially unchanged at [Ca ²⁺]_o up to 1 mM (101.4±7.7%, n=5), but were significa ntly reduced at 2 mM [Ca²⁺]_o (62.0±9.0%, n=5). This reduction at 2 mM [Ca²⁺]_o was abolished by intracellu lar loading of BAPTA (99.6±7.6%, n=6), suggesting that intracellular Ca²⁺ overload, rather than extracellular Ca²⁺ itself, caused the apparent reduction of Mg²⁺ efflux rate. We conclude that the net Mg²⁺ efflux can be driven in the essential absence of intracellular Ca²⁺ and is not modulated by changes in [Ca²⁺]_o. [Jpn J Physiol 55 Suppl:S76 (2005)]

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In animal cells, it is known that under hypoosmotic conditions, an intracellular Ca²⁺ increase initially occurs followed by a regulatory volume decrease (RVD) which is attained by effluxes of K⁺, Cl⁻ and organic osmolytes and the resulting extraction of osmotically obliged water. The present study was performed to identify the volume-regulatory Ca²⁺ influx pathway under hypoosmotic conditions in HeLa cells. Whole-cell recordings showed that osmotic swelling activates TRPM7-like cation currents. The currents showed outward rectification and sharp sensitivity to Mg²⁺, Gd³⁺, SKF96365 and ruthenium red (RR). These TRPM7 channel blockers also inhibited the intracellular Ca²⁺ increase in response to osmotic swelling and the following RVD. RT-PCR studies demonstrated expression of TRPM7 mRNA in HeLa cells. The siRNA silencing of TRPM7 significantly suppressed not only expression of TRPM7 mRNA but also whole-cell TRPM7-like channel currents, Ca²⁺ influx and RVD in HeLa cells upon a hypotonic challenge. Thus, we conclude that the endogenous activity of swelling-activated cation channel exhibits the properties identical to the hallmark biophysical and pharmacological features of TRPM7, and that the TRPM7 channel plays an important role in the RVD process by serving as the volume-regulatory Ca²⁺ influx pathway in the human epithelial cells. [Jpn J Physiol 55 Suppl:S76 (2005)]

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LPA and sphingosine-1-phosphate (S1P) are two major natural bioactive lysophospholipids that induce a wide variety of biological responses mainly via the endothelial differentiation gene (Edg) G protein-coupled receptor family in diverse cell types. Among the Edg family, Edg-1, -3, -5, -6, and -8 (S1P₁–S1P₅) are S1P-specific receptors, and Edg-2, -4, and -7 (LPA₁–LPA₃) are LPA-specific receptors. We previously demonstrated in CHO cells that S1P₁/Edg1 and S1P₃/Edg3 mediated stimulation of Rac and chemotaxis, whereas S1P₂/Edg5 mediated inhibition of a chemoattractant-induced Rac stimulation and chemotaxis. S1P₂ mediated Rho stimulation via G_12/13, and Rho in turn mediated inhibition of Rac and cell migration. Complexly, S1P₃ stimulated Rac and cell migration despite stimulating Rho. Like S1P₃, LPA₁/Edg2, stimulated both Rho and Rac with stimulation of cell migration. The stimulatory effects of both LPA₁ and S1P₃ on Rac and cell migration and Rho are mediated by G_i and G_12/13, respectively. Either Rho inactivation by C3 toxin or inhibition of G_12/13 enhanced LPA₁-mediated stimulation of Rac and cell migration. On the other hand, G_i inactivation by pertussis toxin abolished LPA₁-mediated induced Rac stimulation and cell migration, and induced LPA₁-mediated inhibition of IGF I-induced Rac activation and chemotaxis. These LPA₁-mediated responses were very similar to S1P₃-mediated responses in PTX-treated CHO cells. Thus, LPA₁ generates stimulatory and inhibitory signals via G_i and G_12/13-Rho, which are counteracting with each other, just like S1P₃. [Jpn J Physiol 55 Suppl:S76 (2005)]

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Ghrelin is a peptide isolated from the human and rat stomach as an endogenous ligand for growth hormone secretagoue receptor (GHS-R). mRNAs for ghrelin and GHS-R are present in the pancreatic islets. We previously reported that both the glucose-induced insulin release and cytosolic Ca²⁺ responses to glucose were enhanced by GHS-R antagonist and suppressed by ghrelin in isolated pancreatic islets of the rats. This finding suggested that endogenous ghrelin in islets inhibits glucose-induced insulin release. To further clarify the physiological role of the endogenous ghrelin in insulin release, we employed perfusion system of the isolated rat pancreas that retains the intact microcirculation in islets. Both the first and second phases of glucose-induced insulin release were significantly increased by GHR-antagonist and antiserum against ghrelin, while they were reduced by exogenous ghrelin. However, effects of GHS-R antagonist, anti-ghrelin antiserum and ghrelin on glucose-induced insulin release were absolutely abolished in the rats that were pre-treated with pertussis toxin, a specific inhibitor of certain subtypes of GTP-binding proteins (Gi and Go). These results indicate that endogenous ghrelin inhibits glucose-induced insulin release under physiological conditions preserving microcirculation in islets and that ghrelin exerts the inhibitory effect via GHS-R and in a pertussis toxin-sensitive manner. [Jpn J Physiol 55 Suppl:S76 (2005)]

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C6 glia cells show cell swelling over their initial volume (rebound cell swelling: RCS) at chronic exposure to hypertonic media with sucrose for 48 h. The RCS was accompanied by intracellular vacuolation. RCS was still observed even when we applied blockers of Na⁺/K⁺/2Cl⁻ cotransporter, Na⁺/H⁺ exchanger and Cl⁻/HCO3⁻ exchanger that are generally thought to be involved in regulatory volume increase. On the other hand, RCS or intracellular vacuolation in cells adapting themselves to hypertonic sucrose did not occur in cells treated with a Cl⁻ channel blocker, NPPB. Like NPPB, RCS and intracellular vacuolation were significantly diminished by treatment with inhibitor of stress-activated protein kinase (SAPKs), SB203580 (p38 MAP kinase inhibitor) and SP600125 (JNK inhibitor). These observations suggest that Cl⁻ channel would play a key role in the hypertonicity-induced vacuolation, and that SAPKs including p38 MAP kinase and JNK would be involved in keeping activity of Cl⁻ channel. Taken together the results shown in the present study, we indicate a possibility that the vacuolation could occur in late endosomes and lysosomes, which accumulate H⁺ via vacuolar type H⁺-ATPase into the intra-organella-space coupling with Cl⁻ influx via NPPB-sensitive Cl⁻ channel in a manner dependent on p38 MAP kinase and JNK. [Jpn J Physiol 55 Suppl:S77 (2005)]

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We tested the effects of exposure to a switched 1.5 Tesla magnetic field on transient increase in intracellular Ca²⁺ stimulated by neurotransmitters in bovine adrenal chromaffin cells. The concentration of intracellular Ca²⁺ ([Ca²⁺]i) was increased by addition of acetylcholine (ACh) or bradykinin (BK) in chromaffin cells. These stimulator-induced increase in [Ca²⁺]i in Ca²⁺-free medium was strongly inhibited by 2 hr exposure to the magnetic field, but the exposure did not change BK-induced production of inositol 1,4,5-trisphosphate. In Ca²⁺ containing medium, the exposure delayed only the decay phase of [Ca²⁺]i after peak. Delay of the decay phase was also caused by addition of NaCN in the presence of ACh. The intracellular ATP content and oxygen consumption were influenced by the exposure in glucose-free medium. Measurement of mitochondrial membrane potential by using fluorescent probe, JC-1 (Molecular Probe), indicated depolarization of the membrane in both cells exposed to the magnetic field and added NaCN. These effects of magnetic field would be related to the eddy current. [Jpn J Physiol 55 Suppl:S77 (2005)]

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Cytotoxicity by reactive oxygen species (ROS) are mainly resulted from the reaction with biological molecules. Recently, it was reported that mitochondrial DNA (mtDNA) damage was induced by CoCl₂ through ROS. However, there are only a few reports indicating cytotoxicity of CoCl₂ at the same concentration. Here, we evaluated cell growth, DNA damage and expression of hypoxia inducible factor-1α relevant to the treatment with CoCl₂ in order to determine the responses of 293 cells in detail. For the estimation of DNA damage, we used long PCR method for mtDNA and nuclear-encoded β-globin gene (nDNA). When cells were incubated with 0.4 mM H₂O₂ for 1 hr, mtDNA damage occurred but nDNA remained unchanged. The cell growth was suppressed completely 2 days after the treatment and cell number decreased within 4 days. In the case of CoCl₂ treatment at 100 μM for 24 hr, cell number was the same as that without any treatment throughout the experiment, suggesting that CoCl₂ did not cause cytotoxicity. Furthermore, western blots revealed the activation of hypoxia inducible factor-1α under the conditions. When cells was exposed to 100 μM CoCl₂ for 24 hr prior to 0.4 mM H₂O₂ treatment for 1 hr, we observed a transient growth arrest and the cell number increased after 4days of the treatment of H₂O₂. These data indicate that the pretreatment of CoCl₂ cause the suppression of cytotoxicity by H₂O₂ rather than the induction. [Jpn J Physiol 55 Suppl:S77 (2005)]

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Nitric oxide (NO) generated by neuronal nitric oxide synthase (nNOS) in kidney macula densa (MD) cells functions to vasodilate the afferent arteriole and blunts regulation of tubuloglomerular feedback (TGF). Experiments were performed to examine the effect of NaCl changes in extracellular solution on the NO production from the established mouse macula densa (NE-MD) cells in culture. nNOS protein expression was assessed with Western blotting using the anti-nNOS antibody. Compared with control (normal Ringer solution), nNOS protein levels were time-dependently (0.5, 1, 2, and 5 hrs) increased on the addition of furosemide (loop diureticus). NO release from the NE-MD cells was measured in the presence of 1 mM L-arginine by using the NO sensitive electrode. NO production levels were also time-dependently (2 and 5 hrs) increased on the addition of furosemide, which was dose-dependently (10-50 µM) inhibited by 7-nitroindazole (nNOS specific inhibitor). Similar changes were seen in the NO release when extracellular Cl⁻ concentration was decreased (1/10), but NOT by Na⁺. Furosemide-induced NO generation was completely cancelled when the NE-MD cells were co-incubated with BAPTA-AM (a Ca²⁺ chelator). These results suggest that nNOS expression and NO release in the NE-MD cells is inversely regulated by extracellular Cl⁻ concentration, but NOT by Na⁺ concentration, and that these subcellular mechanisms are sensitive to intracellular Ca²⁺ concentration. [Jpn J Physiol 55 Suppl:S77 (2005)]

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We have previously reported that a macrophage/microglia-specific calcium binding protein Iba1 is involved in advanced motility and phagocytic activity of these cells, and is concerned specifically with signaling of Rac small G protein among members of Rho family GTPases. We report herein identification and characterization of an Iba1 homologous molecule, Iba2, with 63% amino acid identity with Iba1. In contrast to the restricted expression of Iba1 in macrophages/microglia, Iba2 was predominantly expressed in cells in the neural lineage. Iba2 was intensely expressed in the developing brain, of which the expression was localized in nestin-positive cells within the ventricular and subventricular zones, i. e. neural precursor cells. Even in the adult brain, Iba2 was expressed in neural precursor cells in the rostral migratory stream. Furthermore, when Iba2 was expressed simultaneously with dominant active RacV12 in NIH3T3 cells, Iba2 translocated together with F-actin into membrane ruffles induced by RacV12 and the expression of Iba2 potentiated promotion of the membrane ruffling. These results suggest that Iba2 should, as a counterpart of Iba1, participate in a migratory phenotype of neural precursor cells. [Jpn J Physiol 55 Suppl:S78 (2005)]

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Subcutaneously injected, virus-infected Meth A tumor cells grow time-dependently to reach a peak on days 7-12, and are rejected around 3 weeks after injection. However, we reported recently that the rejection, but not the growth, phase was delayed after the specific elimination of macrophages by i.v. injections of dichloromethylene diphosphonate (DMDP)-liposomes. In the present study, we examined the effects of s.c. injections of DMDP-liposomes on the growth of s.c. injected, virus-infected Meth A tumor cells. Of particular interest, the tumor cells were rejected without growth after DMDP-liposome treatment, whereas s.c. injections of PBS-liposomes had no effect on the tumor growth and the rejection. These results suggest that at least two types of macrophages infiltrated into tumor. One infiltrated into tumor at early phase after tumor transplantation to stimulate the growth of virus-infected tumor cells; and the other infiltrated into tumor at late phase after the transplantation to reject the virus-infected tumor cells. [Jpn J Physiol 55 Suppl:S78 (2005)]

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Mitochondria exhibit dynamic movement and morphological changes. We have labeled mitochondria in cultured hippocampal neurons by expressing mitochondria-targeted fluorescent protein and investigated the intracellular signaling that regulates mitochondrial movement and morphology by real-time imaging. Glutamate-induced NMDA dependent Ca²⁺ elevation and calcineurin activation resulted in slow onset of cessation of movement and mitochondrial contraction, which is accompanied by cytochrome c release and neuronal death. High-K⁺ stimulation induced VDCC-dependent Ca²⁺ influx and CaMKI activation. Then, CaMKI phosphorylated dynamin-related protein 1 (Drp1) and induced Drp1 translocation to mitochondria, led to immediate freezing of movement and mitochondrial division, which protected neuron from excitatory toxicity. These results suggest mitochondrial dynamics and morphology are regulated by channel specific intracellular signaling and these changes control neuronal survival and death. [Jpn J Physiol 55 Suppl:S78 (2005)]

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The gastric H ⁺,K⁺–ATPase is involved in the proton secreting mechanism. It is unclear what molecule contributes to the secretion of Cl^–. We previously found that ClC–2 may not be a Cl^– transporting protein for the gastric acid secretion. Here, we examined the expression of another ClC chloride channel, ClC–5, which is abundant in the kidney. Western blot analysis using an anti–ClC–5 antibody showed that ClC–5 is expressed in hog gastric vesicles that are rich in H ⁺,K⁺–ATPase. Double staining with the ClC–5 and H ⁺,K⁺–ATPase antibodies on the hog gastric glands revealed that ClC–5 colocalizes with H ⁺,K⁺–ATPase in the parietal cells. Immunoprecipitation using the anti–H ⁺,K⁺–ATPase antibody demonstrated ClC–5 may bind to H ⁺,K⁺–ATPase in hog gastric vesicles. Furthermore, transient expression of ClC–5 protein in HEK293 cells expressing H ⁺,K⁺–ATPase increased the H ⁺,K⁺–ATPase activity by 20%. ClC–5 is known to be involved in endocytosis in the renal proximal tubular cells where it colocalizes with H ⁺–ATPase. In gastric parietal cells, ClC–5 may be involved in acid secretion in association with H ⁺,K⁺–ATPase. [Jpn J Physiol 55 Suppl:S78 (2005)]

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The function of the two FADs of electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii was investigated. ETF is known to receive electrons from NADH and D-lactate dehydrogenase (LDH) and donate the electrons to butyryl-CoA dehydrogenase. In this study, reactions of ETF with NADH and LDH were analyzed. During NADH titration of ETF, one FAD (named FAD-I) was first reduced in one-electron manner, and the other FAD (FAD-II) was then reduced in two-electron manner. Direct one-electron transfer from NADH to FAD-I is unlikely because NADH is known to be a two-electron reductant. The reduction of FAD-I in one-electron manner can be explained by electron transfer between ETF molecules. This is because the electron transfer from reduced FAD-II to oxidized FAD-I was observed when reduced ETF was mixed with oxidized ETF. LDH reduced only FAD-I in the presence of D-lactate, showing that LDH interacts with FAD-I but not with FAD-II. Fully reduced ETF, where both FAD-I and FAD-II were reduced with NADH, was oxidized by LDH in the presence of pyruvate, resulting in the oxidation of both FAD-I and FAD-II. Kinetic analysis of this reaction revealed that LDH receives electrons from only FAD-I, and the oxidized FAD-I receives electrons from FAD-II. [Jpn J Physiol 55 Suppl:S79 (2005)]

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Protein tyrosine kinases (PTKs) play pivotal roles in numerous cellular functions, i.e., differentiation, proliferation, and morphogenesis, in various types of cells. To reveal the role of PTK in the early preimplantation development, expression and function of PTK in the mouse embryos was examined. In this study, it has been shown that Jak2 is one of the dominantly expressed PTKs in MII stage oocytes. Jak2 protein was localized predominantly in female pronucleus in 1-cell embryos, but not male one. At MII stage and first mitosis, Jak2 was localized to the chromosomes. In culture cells that are known to be express Jak2, localization of Jak2 was detected in the cytoplasm, but not nucleus. Jak2-GFP fusion protein was also localized in cytoplasm of these culture cells, but was in the nucleus of 2-cell embryos. Endogenous Jak2 was not accumulated either in the transplanted nuclei of NIH3T3 or male pronuclei. These results showed that nuclear localization of Jak2 is not common phenomenon in the cells expressing Jak2 and accumulation of Jak2 onto the chromosome is regulated by some factor(s) associated with the chromatins. These results suggest a novel function of Jak2 in the regulation of early preimplantation development. [Jpn J Physiol 55 Suppl:S79 (2005)]

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It has remained unsuccessful to attempt at protein transduction into specific restricted brain areas. We demonstrated the direct in vivo protein transduction by microinjecting β-galactosidase (β-gal) with non-viral vector, hemagglutinating virus of Japan envelope (HVJ-E) vector into the rat nucleus tractus solitarius (NTS). The medulla oblongata including the NTS was removed 6 hr post-injection and cryostat sections were histochemically stained with X-gal to detect β-gal enzymatic activity. In rats microinjected β-gal protein with HVJ-E vector, cells in the area around the injection site were strongly positive for β-gal. The activity of β-gal in sections from rats microinjected with b-gal and HVJ-E vector (102.67±27.64) was significantly (p<0.01) stronger than that of b-galprotein without HVJ-E vector (24.60±8.25). Our findings suggest that direct in vivo protein transduction into specific restricted brain areas is possible. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the central nervous system. [Jpn J Physiol 55 Suppl:S79 (2005)]

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Laminin, an extracellular matrix molecule, is known to promote neurite growth. In the present study, the effects of soluble laminin on organelle transport and their relation to neurite growth were investigated in cultured dissociated mouse dorsal root ganglion (DRG) neurons. Laminin added into the extracellular medium was deposited on the surface of DRG neurons. DRG neurons incubated with soluble laminin exhibited branched, long, and thin neurites. Time-lapse study demonstrated that many small-diameter branches were newly formed after the addition of laminin. Thus, the growths of large-diameter primary neurites arising from cell bodies and branches extended from growth cones of primary neurites were analyzed separately. Laminin decreased the growth rate of primary neurites but increased that of branches. In primary neurites, acute addition of laminin rapidly decreased organelle movement in the neurite shaft and growth cone, accompanied by slowing of the growth cone advance. Branching of primary neurites occurred in response to laminin in some growth cones. In these growth cones, organelles protruded into nascent branches. In branches, soluble laminin increased organelle movement in the growth cone and the distal portion of the shaft. These results suggest that laminin inhibits the elongation of primary neurites but promotes branching and elongation of branches, all of which seem to be closely related to organelle transport. [Jpn J Physiol 55 Suppl:S79 (2005)]

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Ghrelin, isolated from the human and rat stomach, is the endogenous ligand for the growth hormone (GH) secretagogue-receptor (GHS-R). We previously reported that mRNAs for ghrelin and GHS-R as well as immunoreactive ghrelin were detected in the pancreatic islets. This study aimed to explore a physiological role of the islet ghrelin in the regulation of insulin release in rats and to elucidate its action mechanisms in β-cells. GHS-R blockade and anti-ghrelin antiserum markedly enhanced glucose-induced insulin release in isolated islets, while exogenously administered ghrelin suppressed it. In single β-cells, exogenous ghrelin attenuated glucose-induced first phase and oscillatory [Ca²⁺]_i increases via interaction with GHS-R. Ghrelin also increased a tetraethylammonium-sensitive delayed outward K⁺ currents in single β-cells. These effects of endogenous and exogenous ghrelin were not observed in the islets and β-cells following treatment with pertussis toxin (PTX). These findings reveal that endogenous ghrelin in islets acts on β-cells to restrict glucose-induced insulin release via PTX-sensitive mechanisms. [Jpn J Physiol 55 Suppl:S80 (2005)]

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Inward rectifier K⁺ channel(IRK) and G protein-gated inward rectifier K⁺ channel (GIRK) have been recognized as crucial key marker proteins for early differentiation in tunicate embryos, Halocynthias. GIRK is expressed mainly in neural region by in situ hybridization study, while IRK is expressed in epidermal region and suppressed in neural region at the gastrula stage. So, we cloned Halocynthia's TuIRK and TuGIRK genomic genes. Here, we will report phylogenetic analysis of all IRK gene families included in bacterial, fly, worm, and several vertebrate genomic databases. Total 130 IRK family genes were classified into G protein-gated, ATP sensitive, ATP regulated, plain, and bacterial IRK groups. Both tunicate IRK and GIRK amino acid sequences were closely homologous to those of vertebrates. While, Halocynthia IRK and GIRK genomic genes were separated by many introns, some of which sites were observed in the channel pore and the membrane-spanning regions. This feature revealed their marked similarity to those of Ciona and Caenorhabditis. The introns were scarcely found in vertebrate, especially mammalian, IRK and GIRK genomic genes. However, interestingly, exclusively in tunicate and vertebrate GIRK genomic genes, a strongly conserved intron intervening site is found in the region, in which G protein beta likely interacts with GIRK. In addition, there are also two exclusively conserved amino acid sites nearby this region. Altogether, those results suggest that one aspect of phylogenetic evolution in genomic level is closely related to the functional structures of protein. [Jpn J Physiol 55 Suppl:S80 (2005)]

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Hippocalcin is a member of the neuronal calcium sensors (NCS) family predominantly expressed in the hippocampal pyramidal cells. We have found that hippocalcin binds to C-terminal region of mixed leneage kinase (MLK) 3, and inhibits its phosphorylation state. Here we analyzed activity of MLK3 in hippocalcin deficient mice because phosphorylation events in MLK3 play crucial roles in its activation process. Immunoblot analysis using substrate specific anti-phospho-antibody revealed that MKK3, one of the substrate of MLK3, in hippocalcin deficient mice was higher phosphorylated than that in wild type. Kinase activity of immunoprecipitated MLK3 was examined using bacterially expressed MKK4 as a substrate. The resting MLK3 activity in hippocalcin deficient mice was higher than that in wild type. These observations suggest that the binding of hippocalcin to C-terminal domain of MLK3 keeps MLK3 in inactive state via regulating MLK3 phosphorylation state. [Jpn J Physiol 55 Suppl:S80 (2005)]

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To investigate the mechanism of ATP release from the cells by mechanical stimulations, we have developed a real-time ATP imaging system under microscope. Bioluminescence emitted by the reaction of Luciferin-Luciferase and ATP is detected using a high quantam-yield image intensifier and a high performance cooled-CCD camera. The system enables the ATP imaging with 100 ms time-resolution under an upright microscope with x20 water immersion objective (NA0.95). The system also enables simultaneous imaging of Nomarski during ATP luminescence imaging. Using this system we observed [1] touch induced ATP release, [2] stretch induced ATP release, and [3] spontaneous ATP release in Ca²⁺-free solution (CFS), in cultured mammary epithelial cells. In [2], ATP is released intensely from a limited number of cells, and the number increases with increase of stretch length. With a similar manner, in [3], ATP is released, although the time course of the release is completely different. The ATP release by CFS is ten times slower than by stretch, suggesting a difference of ATP releasing mechanism between them. [Jpn J Physiol 55 Suppl:S80 (2005)]

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Background. Although inhibition of angiogenesis represents a beneficial approach for the treatment of various diseases, knowledge about angioinhibitory molecules has been limited. Sphingosine-1-phosphate (S1P) mediates pleiotropic effects in diverse cell types through Edg family receptors. S1P exerts an inhibitory action on cell migration via Edg-5 receptor subtype. We studied whether S1P/Edg-5 system could inhibit angiogenesis. Methods and Results SVEC4-10, a mouse vascular endothelial cell line, possesses the ability to form tube-like structures on the Matrigel. Northern blotting revealed that SVEC4-10 expresses Edg-5 and -3, but not Edg-1. S1P stimulated tube formation and the addition of JTE-013, a specific antagonist against Edg-5, enhanced S1P-induced tube formation. Similarly, JTE-013 potentiated S1P stimulation of cell migration, assessed by a modified Boyden chamber method. In the cells overexpressing Edg-5 (SVEDG5), S1P markedly inhibited epidermal growth factor (EGF)-induced tube formation and migration and the inhibition was reversed by JTE-013. S1P inhibited EGF-induced cellular Rac activation in SVEDG5 cells, and JTE-013 cancelled the S1P inhibition. The Matrigel plug assay in mice revealed that S1P induced angiogenesis in vivo, which was enhanced in the presence of JTE-013. These results suggest that S1P/Edg-5 system plays an inhibitory role in the regulation of angiogenesis, and might be targets of angiostatic and angiogenic therapies. [Jpn J Physiol 55 Suppl:S81 (2005)]

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The rare sugar D-allose, the C-3 epimer of D-glucose, was shown to have a novel inhibitory effect on production of reactive oxygen species (ROS). We also found that D-allose has a novel inhibitory effect on cell proliferation of various human cancer cells. To identify the proteins that may play an important physiological role in apoptosis of cancer cells, we analyzed cancer cells treated with D-allose using a proteomic approach. First, we examined the change of protein phosphorylation after D-allose treatment by Western blotting with anti-phosphotyrosine antibody. Since the various growth factor receptors as well as other intracellular proteins are controlled by phosphorylation, elucidation of tyrosine-phosphorylated proteins may give us clues to understand the mechanism of D-allose-mediated growth inhibition of cancer cells. To analyze the effect of these phosphorylation changes in cancer cells, we compared the global protein expression profile in human cancer cell lines in the presence and absence of D-allose treatment using the stable isotope labeling with amino acids in cell culture (SILAC) method and tandem mass spectrometry (MS/MS). SILAC has recently gained popularity for its ability to compare the expression levels of proteins in a single experiment. Using this method we identified more than two hundred proteins and found some highly up- and down-regulated proteins. On the basis of these findings, we will discuss the possible mechanism of action of D-allose. [Jpn J Physiol 55 Suppl:S81 (2005)]

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The purpose of this experiment and research is to examine what kind of effect reaches the control mechanism of the differentiation of cultured MC3T3-E1 exposed to ELF magnetic fields. Sinusoidal (60Hz) magnetic fields were about 3 mT. Collagen or non collagen protein contents of cultures were measured microscopically by using ImSpector system. In exposure to ELF for 3 days, the treatment of IGF increased in collagen contents for 17 days culture. But the additive effects of the exposure and IGF treatment were not observed. From the experiment using specific inhibitors of PI3 kinase pathways, we obtained the results that these pathways mediated essentially for cell differentiation in response to IGF, but MAP kinase pathways marginally influenced to these differentiation of cells. These data indicated that the signal transduction concerning the collagen synthesis appears in proportion to the different stage of which the cell develop. These results indicate that the mechanisms of differentiation related to IGF in the osteoblasts were altered by the magnetic fields of extremely low frequency. [Jpn J Physiol 55 Suppl:S81 (2005)]

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When exposed to uniaxial cyclic stretch (20%, 1 Hz), cultured human umbilical endothelial cells (HUVECs) gradually change their morphology to complete a spindle-like shape with the major axis perpendicular to the stretch axis within 2hrs. Associated with this response, the level of tyrosine phosphorylation of focal proteins is increased. However, changes in the distribution of tyrosine phosphorylated proteins during stretch, which may relate to the directional shape change, are not known. In this study we measured time-dependent changes in the distribution of tyrosine-phosphorylated focal proteins during uniaxial cyclic stretch. Tyrosine phosphorylated proteins were labeled with fluorescent dye -conjugated PY-20, an antibody against phosphorylated tyrosine. Focal contacts (FCs) of unstretched cells were uniformly stained with PY-20, however, the staining declined in all FCs 2 min after the onset of stretch. Ten min after the stretch, higher level of PY-20 staining was observed at the extending cell edges perpendicular to the stretch axis. These results suggest that uniaxial cyclic stretch induced a transient and global tyrosine dephosphorylation of focal proteins, then caused an stretch-axis dependent tyrosine-phosphorylation of focal proteins. Such a spatially regulated tyrosine phosphorylation may contribute to the directional shape change caused by uniaxial cyclic stretch. [Jpn J Physiol 55 Suppl:S81 (2005)]

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Rare sugars are monosaccharides that exist in a very small amount in nature. Therefore, the physiological functions of rare sugars have not been well studied yet. We have found the inhibitory effect of one of the rare sugars, D-allose, on proliferation of Huh-7 hepatoma cells. Target genes of D-allose were examined using the microarray techniques. Total RNAs purified from the 50 mM D-allose treated or untreated cells were labeled with fluorescent dyes (Cy-3 or Cy-5) and hybridized to the Agilent human 1A microarray containing approximately 20,000 genes. The slide was washed and signals were measured by the microarray scanner. Genes showing a significant expression level change were chosen and further analyzed by realtime PCR method. Our study revealed that the expression of the vitamin D3 up-regulated protein (VDUP), which was reported to induce a cell cycle arrest increased significantly. This increase was specific to the D-allose treatment and was not observed by other sugars such as D-glucose, D-psicose treatment. The MTT assay showed the proliferation inhibiting effect of D-allose on Huh-7 cells and the flow cytometry analysis indicated that D-allose caused the G0/G1 cell cycle arrest. These result suggested that D-allose may inhibit the Huh-7 cell proliferation by activating the VDUP gene pathway. [Jpn J Physiol 55 Suppl:S82 (2005)]

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Cell volume regulation is a fundamental and essential function for mammalian cells to survive. Once cells are exposed to the hypotonic solution, their volume rapidly increases and then gradually returns to the original volume. In the regulatory volume decrease (RVD), swelling-induced activation of the volume-sensitive outwardly rectifying Cl⁻channel (VSOR) plays an essential role. In the present study, we identified ABCF2, which is a member of ABCF subfamily in the ATP-binding cassette (ABC) transporter superfamily, as the VSOR modulator. Overexpression of ABCF2 in HEK293 cells was found to markedly inhibit swelling-induced activation of VSOR currents. Although expression of the ABCF2 transcripts is reported to be ubiquitous, its function has not been known so far. Therefore, this is the first description about a physiological function of ABCF2. In addition, our immunoprecipitation and immunofluorescence studies demonstrated the protein-protein interaction of ABCF2 and actinin-4 (ACTN4). The ABCF2-ACTN4 interaction was dramatically enhanced under hypotonic conditions. Since ACTN4 is an actin-cross-linking protein, it is possible that ACTN4 provides the molecular link between the actin cytoskeleton and VSOR. [Jpn J Physiol 55 Suppl:S82 (2005)]

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Cell migration is a critical component of diverse biological phenomena in both physiological and pathological conditions. Sphingosine-1-phosphate (S1P) has attracted attention as a unique plasma-derived lysophospholipid mediator that either stimulates or inhibits cell migration. Recent studies including our own have disclosed several G protein-coupled receptor (GPCR) subtypes for S1P. These include S1P2/Edg5/AGR16, which we originally cloned as an orphan receptor and identified as the first GPCR that negatively regulates cell migration. Inhibition of a small G protein Rac at the site downstream of Rho activation is a mechanism for S1P2-mediated inhibition of migration. In sharp contrast, S1P1/Edg1 and S1P3/Edg3 mediate activation of Rac and chemotaxis in response to S1P through the Gi-PI 3-kinase pathway. S1P inhibits migration and invasion in vitro of mouse B16 melanoma cells by acting through endogenously expressed S1P2. S1P dose-dependently inhibits melanoma formation after subcutaneous inoculation, and lung metastasis after intravenous injection in vivo, with potentiation of S1P inhibition by S1P2 overexpression. By contrast, overexpression of either S1P1 or S1P3 in B16 cells results in stimulation of migration and invasion in vitro, and aggravation of tumor formation and metastasis in vivo in S1P dependent manners. These results provide the first evidence for GPCR subtype-specific, bi-directional regulation of tumor cell behaviors, raising a novel therapeutic strategy for cancer. [Jpn J Physiol 55 Suppl:S82 (2005)]

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Intrathecal administration of local anesthetics causes neural complications that primarily include sensory defect. In the present study, we observed the micromolphological changes in cultured mouse dorsal root ganglion neurons induced by local anesthetics using video-microscopy. Furthermore, the neurotoxic effects of local anesthetics, lidocaine, tetracaine, procaine, and ropivacaine, were compared. Concentrations used were 5, 12.5, 25, and 50 mM. Molphological changes were observed before and 1, 15, 30, and 60 min after the treatment with local anesthetics. Lidocaine at a concentration of 5 mM had no effect on cell morphology during the observation period. 12.5 and 25 mM lidocaine caused cell swelling 30-60 min after treatment. Treatment with lidocaine at a concentration of 50 mM resulted in membrane rupture within 5 min. Tetracaine and procaine at a concentration of 50 mM caused cell swelling, but did not cause the membrane rupture. Less concentrations of these anesthetics had no effect on cell morphology. Ropivacaine at any concentration had no effect. These results indicate that the order of neurotoxic potency is lidocaine>tetracaine, procaine>ropivacaine, which is similar to the results obtained by clinical studies and in vivo experiments. The Optical investigation may be a simple and useful screening of cytotoxicity of drugs. [Jpn J Physiol 55 Suppl:S82 (2005)]

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Endothelial cells (ECs) sense hemodynamic forces (pressure and shear stress) from the flowing blood. P2X4 is constitutively expressed at high levels in ECs. Yamamoto et al. found a shear stress-dependent Ca²⁺ influx mediated by the purinergic P2X4 receptor as confirmed by sense- and anti-sense oligonucleotide technique. Human embryonic kidney (HEK293) cells that had no Ca²⁺ response to flow, were able to show a cytosolic Ca²⁺ increase when transfected with P2X4. In order to determine if P2X4 can sense the shear force, we investigated whether direct mechanical stimulation of P2X4-containing outside-out patches were activated by flow. Patch-clamp recordings from HEK293 stably expressing of P2X4 showed single channel activity following flow stimulation with Hank's solution. The permeation property of the styryl dye FM1-43 through mechanotransduction channels was also assessed in our case. Epifluorescence and confocal microscopy showed FM1-43(5μM) uptake in HEK293-P2X4 both after ATP (10μM) or flow stimulation. These results indicate functional activity of P2X4 and suggest that P2X4 could likely be a primary shear-stress sensor in endothelial cells. [Jpn J Physiol 55 Suppl:S83 (2005)]

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