Hippocalcin (Hpca) is a member of the neuronal calcium sensor family predominantly expressed in the hippocampal neurons and acts as a multifunctional modulator in the Ca²⁺-signaling underlying memory formation. Here we found several Hpca-associated proteins (HAPs) in the rat brain by filter overlay method using [³⁵S]Hpca. HAP-48, which has a molecular weight of 48kDa showing a Ca²⁺-dependent Hpca-association property, was purified from the cytosolic fraction. Partial amino acid sequences of HAP-48 showed it to be identical to creatine kinase (CK)-B. [³⁵S]Hpca did not associate with CK-M. CK activity of the postmicrosomal fraction from the hippocampus prepared under Ca²⁺-free conditions, was higher than that prepared under Ca²⁺-loaded conditions. The Ca²⁺-dependent translocation of CK activity in the hippocampus was abolished by adding anti-Hpca antibody. In vitro study revealed that myristoylation of Hpca was not required for its association with CK-B, but required for Ca²⁺-dependent membrane transfer of CK-B. The Ca²⁺-dependent membrane transfer of CK-B was diminished in the hippocampus of Hpca-deficient mice. These results indicate that Hpca associates with CK-BB isozyme and transfers it to the plasma membrane in a Ca²⁺-dependent manner, suggesting that Hpca plays an important role in local energy shuttling system. [J Physiol Sci. 2008;58 Suppl:S203]

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It has been established that intra- and extracellular mechanical environments affect the 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 the development of FAs. Therefore, we examined the effects of mechanical forces on actin polymerization at FAs. When cell contractility was inhibited by the myosin II inhibitor blebbistatin, actin polymerization at FAs was diminished. By contrast, when blebbistatin-treated cells were subjected to a sustained uniaxial stretch, the actin polymerization was restored. These results demonstrate the novel role of mechanical forces in actin polymerization at FAs. To reveal the molecular mechanism underlying the force-induced actin polymerization at FAs, we examined the distribution of zyxin, a postulated actin-regulatory protein. Actin polymerizing activity was strong at FAs with large amounts of zyxin. Accumulation of zyxin at FAs was diminished by blebbistatin, but was restored by a uniaxial stretch. When endogenous zyxin was displaced from FAs by expressing the FA-targeting region of zyxin, the force-induced actin polymerization at FAs was decreased. These results provide direct evidence that zyxin is involved in the mechanical force-dependent facilitation of actin polymerization at FAs. [J Physiol Sci. 2008;58 Suppl:S203]

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The Rho-kinase (ROK)-mediated Ca²⁺-sensitization of VSM contraction contributes to abnormal VSM contraction such as vasospasm. As upstream messengers of ROK, we have identified SPC and Fyn tyrosine kinase. In contrast, as the downstream pathways, the ROK-mediated elevation of myosin light chain (MLC) phosphorylation was well documented, and it has been hypothesized that ROK not only directly phosphorylates MLC, but also indirectly increases MLC phosphorylation through the inhibition of myosin phosphatase. Although several molecules are proposed to mediate these downstream pathways of ROK, essential molecule(s) have not been determined yet. In order to clarify this point, in this study we used in vitro motility assay system with highly purified contractile proteins of smooth muscle, but in the complete absence of the MLC kinase and phosphatase. The chicken gizzard regulatory and essential MLCs and heavy meromyosin (HMM) were purified as recombinant proteins in a baculovirus system and were confirmed by MALDI-TOF MS. The HMM was phosphorylated by ROK, as confirmed by urea-glycerol PAGE. The in vitro motility assay of these purified proteins revealed that average velocity of actin sliding on unphosphorylated and ROK-phosphorylated HMM was 0.0 and 0.3 μm/sec, respectively, the latter of which is comparable with the maximum values of MLCK-phosphorylated ones (0.27 μm/sec). [J Physiol Sci. 2008;58 Suppl:S204]

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Hypercholesterolemia is a major risk factor of cardiovascular events. A Rho-kinase (ROK)-mediated Ca²⁺ sensitization of vascular smooth muscle (VSM) plays a critical role in abnormal VSM contraction such as vasospasm. We found that SPC activates this pathway, dependently on cholesterol in the VSM tissues and cells and through the activation of Fyn. These results, taken together with the localization of Fyn in cholesterol-enriched membrane microdomains, membrane lipid rafts, suggest the essential roles of membrane rafts in abnormal VSM contraction. Although several signaling molecules, such as receptors or kinases, accumulate membrane rafts, the membrane-permeabilization of VSM revealed that the activation of G-proteins or GPCRs was not required for the SPC-induced contraction. Therefore, we aimed to investigate the possible interaction of SPC with membrane lipid rafts. For this purpose, we successfully and originally developed the model membrane lipid rafts, which contain high concentrations of cholesterol and sphingomyelin, and examined their direct interaction with SPC, using the surface plasmon resonance measurement (BIACORE system). Here we provide the first direct evidence that SPC has very high affinity for the model membrane lipid rafts, but very weak affinity for the normal control model membrane, containing mainly phosphatidylcholine. This notion also supports the important roles of cholesterol and its enriched membrane, lipid rafts. [J Physiol Sci. 2008;58 Suppl:S204]

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We studied a role of p38 MAPK in the chronic hypotonicity-stimulated transepithelial Na⁺ reabsorption in renal epithelial A6 cells. Pretreatment with SB202190 (a specific p38 MAPK inhibitor) drastically decreased the chronic hypotonicity-stimulated transepithelial Na⁺ reabsorption by reducing the apical Na⁺ entry through epithelial Na⁺ channel (ENaC) and the besolateral Na⁺ extrusion via the Na⁺/K⁺ ATPase (pump), although the rate limiting step was still the Na⁺ entry step. We further examined whether the inhibitory effects of SB202190 on the transepithelial Na⁺ reabsorption is caused through suppression of mRNA expression of ENaC participating in the transepithelial Na⁺ reabsorption as the Na⁺ entry pathway. The chronic hypotonicity increased the mRNA expression of alpha-, beta-, and gamma-subunits of ENaC. Moreover, we found that inhibition of p38 MAPK by SB202190 suppressed the mRNA expression of beta- and gamma-ENaC but not alpha-ENaC. Based on these results, it is suggested that the chronic hypotonicity stimulates the Na⁺ reabsorption by upregulating the beta- and gamma-ENaC mRNA expression via a p38MAPK-dependent pathway. This work was supported by Grants-in-Aid from Japan Society of The Promotion of Science (17390057, 17590191,18659056, and 19590212), Fuji Foundation for Protein Research, and The Salt Science Research Foundation (0736). [J Physiol Sci. 2008;58 Suppl:S204]

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We previously reported the nongenomic mechanism of hypotonicity-induced renal Na⁺ reabsorption which involves protein tyrosine kinase (PTK) activation. However, it is still unknown what type of PTK mediates this stimulation. In the present study, we investigated the role of receptor-type tyrosine kinase (RTK) on the hypotonic stimulation of Na⁺ transport. In monolayers of A6 cells, a renal cell line derived from Xenopus laevis, we observed inhibitory effects of AG1478 (an inhibitor of EGF receptor) and AG1296 (an inhibitor of PDGF receptor) on both the hypotonicity-induced stimulation of Na⁺ transport and the hypotonicity-induced ligand-independent activation of EGFR. Next, we investigated a linkage between RTK and MAP kinase family members, indicating that ERK1/2 and JNK, but not p38 MAPK, are activated in the downstream of RTK and only JNK was involved in the hypotonic induction of Na⁺ transport. A further study suggests that RTK-JNK cascade contributes to the protein synthesis-independent early phase in the hypotonicity-induced Na⁺ absorption, but not to the protein synthesis-dependent late phase. Further, the present study suggests involvement of phosphatidylinositol 3-kinase in RTK-JNK cascade-mediated Na⁺ transport. These observations indicate that: 1) hypotonic stress activates JNK via RTK through a ligand-independent pathway, 2) the RTK-JNK cascade acts as a mediator of the hypotonic stress for stimulation of Na⁺ transport, and 3) PI3-kinase is involved in the RTK-JNK cascade to hypotonic stress-induced stimulation of Na⁺ transport. [J Physiol Sci. 2008;58 Suppl:S204]

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The Na⁺/K⁺/2Cl⁻ cotransporter isoform 1 (NKCC1) mediates electroneutral transport of Na⁺, K⁺ and 2Cl⁻ into the cell, and plays crucial roles in homeostasis of cellular Cl⁻ content and cell volume regulation. Turnover of many protein is mediated by covalent attachment of the small protein ubiquitin. Polyubiquitinated proteins are degraded by 26S proteasome. Monoubiquitinated plasma membrane proteins undergo endocytosis and degraded in lysosome, although polyubiquitination is involved in degradation of plasma membrane proteins in some case. We previously demonstrated that NKCC1 is essential for the NGF-induced neurite outgrowth in rat pheochromocytoma PC12 and PC12D cells. The aim of this study is to declare 1) which pathway, or both pathways, is responsible for the degradation of NKCC1 protein, and 2) whether the rate of neurite elongation has correlation with the protein level of NKCC1. Using western blotting and microscopy technique, we found that: 1) the protein level of NKCC1 and the neurite elongation rate in PC12D cells were much higher than those in PC12 cells, 2) the degradation of NKCC1 protein in PC12D cells was much slower than that in PC12 cells, and 3) the degradation rate of NKCC1 in PC12 cells was diminished by treatment with MG132 or clasto-lactacystin β-lactone (a proteasomal inhibitor), but not with chloroquine (a lysosomal inhibitor). These results suggest that the rate of degradation and turnover of NKCC1 protein is mainly mediated by the proteasomal system, and that the high expression level of NKCC1 in PC12D cells is due to a slow degradation rate compared with PC12 cells. [J Physiol Sci. 2008;58 Suppl:S205]

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Interleukin-1β (IL-1β) influences cell injury in various organs during inflammatory diseases. Although some investigators reported that changes in K⁺ channel activity were involved in the renal tubular cell injury induced by endotoxemia or ischemia, little information is available regarding effects of cytokines on activity of renal K⁺ channels. In this study, we investigated the effects of IL-1β on activity of an inwardly rectifying K⁺ channel (Gi: 40 pS, Go: 7 pS) in cultured human proximal tubule cells, using the cell-attached mode of patch-clamp technique. IL-1β (15 pg/ml) reversibly suppressed the K⁺ channel activity in a few minutes after its addition to the bath. This acute suppressive effect was blocked by IL-1 receptor antagonist (20 ng/ml). 8Br-cAMP (100 μM) or 8Br-cGMP (100 μM) did not stimulate channel activity in the presence of the suppressive effect of IL-1β. A PKC inhibitor, GF109203X (500 nM), reactivated the channel suppressed by IL-1β. Furthermore, pretreatment of cells with GF109203X prevented the suppression of channel activity by IL-1β. These results suggested that IL-1β acutely suppressed activity of the 40 pS K⁺ channel in cultured human proximal tubule cells by impairing the PKA/PKG pathways and activating PKC-mediated phosphorylation processes. [J Physiol Sci. 2008;58 Suppl:S205]

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Previous study indicated that activation of adrenergic α₁ receptor in urothelium induced ATP release, which was thought as a trigger of micturition reflex. However, the relevance of α₁ receptor in urothelium to the structure and the motility is remained to be elucidated. Here we explored the influence of activation of α₁ receptor on morphology and motility in urothelium using primary cell culture system. The role of α₁ receptor was estimated with cell staining, time lapse analysis and wound healing assay. High dose of phenylephrine (10μM) rapidly induced morphological change in urothelium, especially in the peripheral region formed with pseudopodia and lamellipodia, revealed by F-action and paxillin staining. From the time lapse analysis, the motility was significantly increased by phenylephrine (10nM), while it was not significantly increased by isoproterenol (10nM). The healing from the wound was dose-dependently ameliorated by the phenylephrine (0.01 nM to 10 nM). The amelioration induced by phenylephrine was inhibited by the addition of α₁ non-selective-antagonist, prazocin (1 nM), and α_1A selective-antagonist, silodosin (1 nM). The density of pseudopodia and nucleus on the wounded edge was significantly increased after phenylephrine treatment. Both responses were inhibited by pretreatment of silodosin (1 nM). These results indicated that activation of α_1A receptor caused the proliferation and the motility of urothelium on wound healing process through morphological change. The pathophysiological process of neurogenic cystitis might be depend on α_1A receptor of urothelium. [J Physiol Sci. 2008;58 Suppl:S205]

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Reactive oxygen species (ROS) are known to cause neurodegenerative diseases. We have previously shown that ROS impair axonal transport. In particular, hydrogen peroxide (H₂O₂) has remarkable toxicity for neuronal cells. In the present study, we investigated the effects of H₂O₂ on neuronal cells in cultured mouse dorsal root ganglion neurons. Cell membrane disruption was evaluated by trypan blue staining. Mitochondria and Lysosomes were stained with Mitotracker and Lysotracker, respectively, in living cells. Cytoskeletons, actin filaments and microtubules, were stained with rhodamine–phalloidin and anti -α–tubulin antibody, respectively, after cells were fixed with 4% paraformaldehyde. Organelles and cytoskeletons were observed under fluorescence microscopy. Immediately after treatment with tert-butyl hydroperoxide (tBHP, 1 mM), a H₂O₂ donor, cell bodies as well as axons were stained by trypan blue. Mitochondria and lysosomes were swollen within 2 min after treatment. In contrast, actin filaments and microtubules were not apparently changed. These results indicate that cell membrane and intracellular membranous structures may be the target of H₂O₂. This may lead to irreversible axonal degeneration and neuronal cell death. [J Physiol Sci. 2008;58 Suppl:S205]

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Melanosomes increases in melanocytes in response to the DMSO or the anticancer agents. This process is considered as the cytoprotective mechanisms against the cytotoxic chemicals or reactive oxygen spices because such agents can be captured by melanosomes and excluded by the exocytotic process.We already reported that the melanogenesis increased in B16 melanoma cells in thelow-glucose culture media without the cell damage. In this study we analyzed the responses of B16 cells against different types of cytotoxic stress in the low-glucose culture media and found that Hypoxia-inducible factor-1 (HIF-1) was induced in the low-glucose conditions by RT-PCR methods and the resistance against the hydrogen peroxide increased in comparison with the normal-glucose conditions. [J Physiol Sci. 2008;58 Suppl:S206]

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Recently, we identified a novel receptor-independent G-protein activator, Activator of G-protein signaling 8 (AGS8), from cDNA library of rat hearts subjected to repetitive transient ischemia. AGS8 was induced in the ischemic myocardium and regulated Gβγ signaling. In face of myocardial ischemia and/or ischemia/reperfusion, AGS8 would provide an additional signal input responsible for myocardial adaptation, however the nature of this protein has not been characterized. As an initial step to investigate physiological roles of AGS8, we examined an impact of AGS8 on hypoxia-induced apoptosis in cardiomyocyte and its subcellular localization. Cultured rat neonatal cardiomyocyte (NCM) were exposed to hypoxia for 6, 24 hr or hypoxia (6hr)/reoxygenation (18hr) following treatment of siRNA for AGS8. Apoptosis of cells were determined by DNA end-labeling, annexin V, and/or mitochondrial membrane potential. Surprisingly, silencing of AGS8 inhibited apoptosis of cardiomyocyte induced by hypoxia alone as well as hypoxia/reoxygenation. Antibody against AGS8 recognized immunoreactive signals that enriched at cell-cell interface. These data suggested that AGS8 was involved in the formation of apoptotic signaling induced by hypoxia and that AGS8-enriched spots might be contributed to an integration of such signals. An effective inhibition of apoptosis by siRNA for AGS8 implies a potential of therapeutic target to protect the myocardium from ischemic events. [J Physiol Sci. 2008;58 Suppl:S206]

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Cancer cells responsive to treatment with cisplatin, an anti-cancer drug, undergo apoptosis. To investigate the possible role of cation channels in the cellular response to cisplatin, we examined the activity of cation channels in cisplatin-sensitive KB-3-1 (KB) epidermoid cancer cells by the whole-cell patch-clamp method. Activity of the Ca²⁺-activated, intermediate conductance K⁺ channel (IK1) was observed during hypotonic stimulation of these cells. The channel was inhibited by the blockers clotrimazole and TRAM-34. In addition, expression of a dominant negative construct suppressed its activity. In the KCP-4 cell line, a cisplatin-resistant cell line derived from KB cells, we did not observe IK1 activity, and the channel's molecular expression, observed by semi-quantitative RT-PCR and immunostaining, appeared much reduced. To determine whether IK1 has a physiological role as part of the volume regulatory machinery in KB cells, cell volume measurements were made; it was found that regulatory volume decrease during hypotonic stress was inhibited by IK1 blockers, implicating the channel in this process. Next, we examined whether the channel has a role in the cisplatin-induced apoptosis of KB cells. It was found that IK1 blockers inhibited a cell viability decrease, as well as a caspase-3/7 activity increase, caused by cisplatin treatment. Thus, the activity of IK1, functional in cisplatin-sensitive KB cells but not in cisplatin-resistant KCP-4 cells, appears to contribute to apoptotic cell death in cisplatin-treated KB cells. [J Physiol Sci. 2008;58 Suppl:S206]

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Rare sugars are defined as monosaccharides that exist in nature but are only present in limited quantities. We examined the effect of D-allose on the proliferation of cancer cells and the underlying molecular mechanism of the action. The HuH-7 hepatocellular carcinoma cells were treated with various monosaccharides for 48 hrs and D-allose was shown to inhibit cell growth by 40% in a dose dependent manner. D-allose induced the G1 cell cycle arrest but not apoptosis. The microarray analysis revealed that D-allose significantly up-regulated thioredoxin interacting protein (TXNIP) gene expression, which is often suppressed in tumor cells, and western blot analysis confirmed its increase in protein level. The overexpression of TXNIP induced G1 cell cycle arrest, too. Analysis of cell cycle regulatory genes showed p27kip1, a key regulator of G1/S cell cycle transition, to be increased at the protein but not the transcriptional level. Protein interaction between TXNIP and jab1, and p27kip1 and jab1, was observed, suggesting stabilization of p27kip1 protein by the competitive inhibition of jab1-mediated nuclear export of p27kip1 by TXNIP. In addition, increased interaction and nuclear localization of TXNIP and p27kip1 were apparent after D-allose treatment. Our findings surprisingly suggest that D-allose, a simple monosaccharide, may act as a novel anticancer agent via unique TXNIP induction and p27kip1 protein stabilization. [J Physiol Sci. 2008;58 Suppl:S206]

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Growth of prostatic epithelial cells is androgen-dependent, however the mechanism of androgen action on the progression of cell cycle is not fully defined. We investigated whether the androgen-dependent prostatic epithelial cell growth is mediated by androgen regulation of expression and activation of cell cycle associated proteins. For this purpose, we used androgen-dependent prostatic cancer cell line, LNCaP-FGC. We found that the proliferation of LNCaP cell was significantly arrested in G₁/S and G₂/M phases after treatment with the charcoal-dextran stripped serum-containing medium. Dehydrotestosterone (DHT) at low concentrations (0.001 to 0.1 nM) stimulates cell growth dose-dependently. However, DHT at high concentrations (1.0 to 100 nM) inhibits cell proliferation by arresting the cell cycle only in the G₁ phase. To clarify the molecular mechanisms causing these differences, we studied the influence of DHT on several cell cycle signaling molecules. The effects of DHT on major cell-cycle regulators such as cyclin-dependent kinase inhibitors (CDIs), cyclin-dependent kinases (CDKs) and their downstream signaling molecules will be discussed. [J Physiol Sci. 2008;58 Suppl:S207]

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Modulation of ion permeability of the plasma membrane during the cell cycle is one of the key events in cell cycle progression. Our recent studies indicate that the intracellular chloride could act as a signal to regulate important cellular functions. The aim of the present study was to investigate whether the intracellular chloride affects cell growth and cell cycle progression of cancer cells. In human gastric cancer MKN28 cells, culture in the Cl⁻-replaced medium (replacement of NaCl by NaNO₃) induced a significant decrease of the intracellular chloride concentration ([Cl⁻]_i). A cell proliferation assay indicates that cell growth was significantly inhibited by the exposure of cells to the Cl⁻-replaced medium. We found that cells cultured in Cl⁻-replaced media showed cell cycle arrest at the G₀/G₁ phase, and that CDK2 and phosphorylated Rb were decreased in low Cl⁻-treated cells. Further analysis revealed that the exposure to Cl⁻-replaced media significantly increased expressions of p21 CDK inhibitor protein in a p53-independent manner. These findings suggest the regulation of [Cl⁻]_i is a novel, unique therapeutic strategy for gastric cancer treatment. [J Physiol Sci. 2008;58 Suppl:S207]

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Our recent studies demonstrate that the reduction of intracellular Cl⁻ concentration bring the inhibitory effect on the cell proliferation of MKN28 gastric cancer cell by diminishing the transition rate from G₁ to S cell cycle phase through a p21, CDK inhibitor, upregulation in a p53-independen manner. However, it is still unknown how the intracellular Cl⁻ regulates the p21 expression level. In this study, we show that stress-activated protein kinases (SAPKs) are involved in p21 upregulation and cell cycle arrest induced by the reduction of the intracellular Cl⁻ concentration. In MKN28 cells, culture in the Cl⁻-replaced medium (replacement of NaCl by NaNO3) significantly induced SAPKs (ERK, p38, and JNK) phosphorylation and G₁/S cell cycle arrest. To clarify the involvement of SAPKs in p21 induction and cell growth inhibition in the Cl⁻-replaced medium, we studied the effects of specific SAPKs inhibitors on the p21-induced G₁/S cell cycle arrest in MKN28 cells. The cell cycle arrest observed in the Cl⁻-replaced media was partially eliminated by the addition of inhibitors of SAPKs. The effects of SPAKs inhibitors on the p21 and its downstream signaling molecules will also be discussed. [J Physiol Sci. 2008;58 Suppl:S207]

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We showed ion replacement of extracelluar fluids could effect ion flow through lysosomal membrane, as a result, it suppressed necrotic cell death caused by amphotericin B. Melanosome and lysosome share intravacuolar acidity, membrane protein and ion channel such V-ATPase, because both of them are originated from late endosomes. Bafilomycin A1, a V-ATPase blocker, induces tyrosinase activity and melanosome maturation with pH increase. We analyzed melanogenesis after ion replacement with/without bafilomycin A1 in this study. [J Physiol Sci. 2008;58 Suppl:S207]

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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 regulating mechanism is unknown. In the previous meeting, we reported that the interaction between a cytosolic ABC protein member, ABCF2, and an actin-binding protein, α-actinin 4 (ACTN4), is involved in the cell volume regulation after osmotic swelling, and that ABCF2 is a downregulator for activation of the whole-cell VSOR current. Here we introduced discrete mutations into the nucleotide-binding domain (NBD) of ABCF2, G232D in NBD1 and G520D in NBD2, and investigated its effects on the VSOR current and the interaction with ACTN4. Whole-cell patch-clamp experiments showed that ABCF2-G520D completely, whereas ABCF2-G232D partially, lost its negative effects on the VSOR current. Immunoprecipitation studies demonstrated that ABCF2-G520D can interact with ACTN4 as effectively as ABCF2-WT under hypotonic conditions, whereas ABCF2-G232D exhibits a higher affinity to ACTN4 than ABCF2-WT. Thus, it is suggested that the VSOR-regulating effect of ABCF2 requires the intact conformation of its NBD2 and depends on the proper interaction between ABCF2 and ACTN4. [J Physiol Sci. 2008;58 Suppl:S208]

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The maxi-anion channel has been suggested to represent a major ATP-conductive pathway mediating swelling-induced ATP release from many cell types. At the previous meeting, we reported that the channel is kept in the closed state while tyrosine-phosphorylated, whereas the dephosphorylation favours channel opening. However, the available spectrum of blockers for protein tyrosine phosphatases did not allow precise identification of this particular type of phosphatase. In the present study, we tested a possible involvement of the cloned receptor tyrosine phosphatase, RPTPζ, in the channel activation mechanism using molecular biological approaches. The rate of excision-induced maxi-anion channel activation was significantly slower in mouse adult fibroblasts (MAFs) derived from RPTPζ-deficient mice compared to MAFs from wild-type (WT) mice. Transfection of RPTPζ, but not the dominant-negative mutant, into the MAFs from RPTPζ-KO mice restored the maxi-anion channel activation rate to the level comparable to that of the WT MAFs. These results strongly suggest that RPTPζ represents an important part of the activation mechanism of the maxi-anion channel. The time-dependent ATP release from swollen MAFs derived from RPTPζ-KO mice was significantly lower compared to that from WT, suggesting that RPTPζ is involved in the swelling-induced release of ATP possibly via maxi-anion channel activation. [J Physiol Sci. 2008;58 Suppl:S208]

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The volume-sensitive outwardly rectifying (VSOR) chloride channel is ubiquitously expressed and involved in cell volume regulation after osmotic swelling in various cell types. In adipose cells, we previously found that a preadipose cell line 3T3-L1 expresses functional VSOR channels. Here, by using a whole-cell patch-clamp technique, we examined whether or not the VSOR channel is expressed in white adipocytes acutely isolated from mice epididymal fat pads. Whole-cell voltage-clamp recordings revealed that chloride currents were gradually activated upon cell swelling induced by application of hypotonic solution. This current was inhibited by a chloride channel blocker, NPPB, or hypertonic solution, and showed outward rectification and inactivation kinetics at large positive potentials. These electrophysiological and pharmacological properties are consistent with those of the VSOR channel in other cell types. It is concluded that the VSOR channel is functionally expressed in white adipocytes acutely isolated from mice. [J Physiol Sci. 2008;58 Suppl:S208]

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Insulin stimulates glucose uptake by adipocytes, whereas TNFα impairs this insulin sensitivity thereby inducing insulin resistance in the cells. Since insulin and TNFα have been shown to induce cell swelling and shrinkage, respectively, in many other cell types, there is a possibility that a change of cell volume serves as a signal for insulin sensitivity and insulin resistance in adipocytes. To test this possibility, the present study was performed in 3T3-L1 adipocytes. Cell size measurements showed that insulin induced swelling and this insulin-induced swelling was inhibited by addition of a blocker of Na⁺/H⁺ exchanger, amiloride, or that of anion exchanger, DIDS. Amiloride or DIDS also inhibited insulin-induced translocation of GLUT4 to the plasma membrane and glucose uptake. TNFα suppressed insulin-induced GLUT4 translocation, glucose uptake and cell swelling, and these TNFα effects were inhibited by a Cl⁻ channel blocker, NPPB or glibenclamide. Whole-cell patch clamp studies demonstrated that TNFα activated volume-sensitive outwardly rectifying (VSOR) Cl⁻ currents. TNFα also induced cell shrinkage in a manner sensitive to NPPB or glibenclamide. Taken together, it appears that cell volume changes are involved in the mechanisms of insulin sensitivity and TNFα-induced insulin resistance in adipocytes. [J Physiol Sci. 2008;58 Suppl:S208]

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We tested effects of exposure to a switched 1.5 Tesla magnetic field on actin protein during cell volume regulation in hypotonic medium in cultured bovine adrenal chromaffin cells. Cellular G-actin was increased by the exposure or addition of cytochalasin D, and addition of jasplakinolide to 2 hr-exposed cells decreased G-actin and maintained the normal value. Intracellular morphology and distribution of F-actin measured by confocal laser scanning microscope were changed, but jasplakinolide did not recover the changes. The cell volume was increased rapidly after incubation in hyposmotic medium, followed by their gradual decrease. This cell volume was almost restored to their normal level. Exposure to the time-varying magnetic field increased the peak value and delayed the recovery to the normal value. Preincubation of cytochalasin D indicated the same volume changes as 2 hr-exposed cells. Replacement to hyposmotic medium caused transient decrease in cellular F-actin and then the content was increased instantly to the normal value within 30 min in control cells. These data are suggested that exposure to the time-varying magnetic field influence the volume regulation by affecting actin polymerization or depolymerization. [J Physiol Sci. 2008;58 Suppl:S209]

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Cell shrinkage, called apoptotic volume decrease (AVD), is one of the characteristic early events in the apoptosis processes. On the other hand, the hypertonicity-induced cation channel (HICC) plays a highly efficient role in the regulatory volume increase (RVI) of many cell types after osmotic shrinkage. By means of electronic cell sizing and whole-cell patch-clamp techniques, in the present study, effects of HICC activation on the staurosporine (STS)-induced AVD and apoptosis were examined in HeLa cells on the basis of a differential time protocol for activation of both processes and by use of blockers of the HICC. Hypertonic stress was found to reduce STS-induced AVD, cell death, caspases 3/7 activation and DNA fragmentation, and these effects were actually due to activation of the HICC. On the other hand, treatment with STS was found to significantly diminish osmotic HICC activation. Thus, it is concluded that the HICC plays not only a volume-restoring role in osmotic shrunken cells but also a death-rescuing role in apoptotic shrunken cells. [J Physiol Sci. 2008;58 Suppl:S209]

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Effects of metabolic inhibition and intracellular pH on the Na⁺-dependent Mg²⁺ efflux in rat ventricular myocytesMichiko, Tashiro; Masato, Konishi (Dept. physiol., Tokyo Med. Univ., Tokyo, Japan)We measured cytoplasmic Mg²⁺ concentration ([Mg²⁺]_i) with the fluorescent indicator furaptra under Ca²⁺-free conditions. Treatment of the cells with 1 μM FCCP (10-15 min) or 5 mM KCN (60-90 min) in the absence of extracellular Na⁺ significantly increased [Mg²⁺]_i from ∼1 mM to ∼2.5 mM. The Na⁺-dependent Mg²⁺ efflux activity was estimated from the initial rate of in [Mg²⁺]_i (initial Δ [Mg²⁺]_i/Δt) for 30-150 s after introduction of extracellular Na⁺. The Mg²⁺ efflux activity was largely reduced, on average, by 87.6%(n=8) in FCCP-treated cells and 90.5% (n=4) in KCN-treated cells, compared with that in intact cells with comparable [Mg²⁺]_i. [Na⁺]_i measured with a Na⁺indicator SBFI was, on average, 5.0-10.5 mM (n=4) within the time range for initial Δ[Mg²⁺]_i/Δt measurements, which is much lower than that required for 50% inhibition of the Mg²⁺ efflux (–40 mM). Clamping intracellular pH to 7.15 by application of nigericin did not reverse the inhibition of the Mg²⁺ efflux. In intact cells, intracellular alkalosis upon application of 20 mM NH₄Cl did not significantly change, while intracellular acidosis upon removal of NH₄Cl significantly reduced the Mg²⁺ efflux, on average, by 54.5% (n=4). These results suggest that the Na⁺-dependent Mg²⁺ efflux critically requires cellular metabolism, although intracellular acidosis caused by metabolic inhibition may also contribute partly to the inhibition of the Mg²⁺ efflux. [J Physiol Sci. 2008;58 Suppl:S209]

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Electroneutral NaCl absorption in the colon is mediated by functional coupling of the Na⁺/H⁺ exchanger and Cl⁻/HCO₃⁻ exchanger. SLC26A3, Cl⁻/HCO₃⁻ exchanger, is highly expressed in colonic epithelial cells, and mutations in the SLC26A3 cause congenital chloride diarrhea. These suggest that SLC26A3 plays the role in NaCl absorption in the colon. It is proposed that the coupling of these exchangers may occur as a result of indirect linkage by changes of intracellular pH (pHi). We therefore investigate whether SLC26A3 is regulated by intracellular pH. We generated an N-terminal hemagglutinin epitope-tagged human SLC26A3 construct and expressed transiently in CHO cells by using inducible gene expression systems. Transport activities were measured with a fluorescent pH-sensitive dye, BCECF and a chloride-sensitive dye, MEQ. pHi was clamped at a range of value from 6.0 to 7.4 with varying K⁺ concentrations of medium that contained the K⁺/H⁺ exchanging ionophore, nigericin. Since SLC26A3 transports nitrate, we monitored changes of intracellular Cl⁻ as chloride exchanges for nitrate and also as chloride exchanges for bicarbonate at varying pHi. Whereas Cl⁻/HCO₃⁻ exchange was steeply inhibited by acidic pHi, Cl⁻/NO₃⁻ exchange was essentially insensitive to pHi. We also characterized pHi dependency with the entire C-terminal truncation mutant, delta 524. Removal of C-terminal decreased activity, but did not noticeably affect the inhibition by intracellular acidification. These results suggest that cytoplasmic domain of c-terminus does not contribute to regulation of SLC26A3 by pHi. [J Physiol Sci. 2008;58 Suppl:S209]

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The electrogenic Na⁺-HCO₃⁻ cotransporter (NBCe) plays a critical role in secretion and absorption of HCO₃⁻ and regulation of intracellular pH in various tissues, and thus its activity is necessary to be regulated tightly. Despite intensive studies, the regulatory mechanisms of NBCe activity have not been fully understood. It has been shown that intracellular Mg²⁺ (Mg²⁺_i) regulates various ion channels and transporters in several ways. In this presentation, we show using the standard whole-cell patch clamp technique that Mg²⁺_i can control natively and heterologously expressed NBCe currents. Mg²⁺_i concentration was varied by pipette solutions having different calculated free Mg²⁺. Native NBCe currents recorded from freshly dissociated bovine parotid acinar cells were inhibited by increasing Mg²⁺_i in a dose-dependent manner (K_i =–1 mM). The native currents were also inhibited by intracellular polyvalent cationic compounds such as neomycin and spermine. Mg²⁺_i and neomycin inhibited heterologous NBCe currents in mammalian cultured cells transiently transfected with the cloned bovine parotid NBCe1-B. These results suggest that Mg²⁺_i might regulate the NBCe1-B activity under physiological conditions, and the underlying mechanism will be discussed. [J Physiol Sci. 2008;58 Suppl:S210]

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The ductal system of the exocrine pancreas produces a HCO₃⁻-rich fluid secretion in response to secretin and some other simuli. Basolateral K⁺ channels provide an exit pathway for K⁺ brought in by the basolateral Na⁺-K⁺-ATPase and play a vital role in maintaining the membrane potential, which is a crucial component of the driving force for anion secretion. Using electrophysiological and molecular techniques, we investigated molecular nature of K⁺ channels which contribute to the resting membrane potential in pancreatic duct cells. In whole-cell recordings from freshly isolated interlobular duct cells, we observed a strongly inwardly rectifying K⁺ (Kir) current, which displayed an approximate square-root dependence of conductance on extracellular K⁺ concentration and a high degree of selectivity for K⁺. The currents were blocked by extracellular Ba²⁺ in a voltage-dependent manner. Cell-attached single-channel recording identified 40 pS K⁺ channels that are most likely to mediate whole-cell Kir currents. RT-PCR of RNA from rat pancreas revealed transcripts of rat Kir2.1, Kir2.2, and Kir2.4. Immunohistochemical studies showed Kir2.1, Kir2.2 and Kir2.4 immunoreactivity in pancreatic duct cells. These results suggest that Kir2 subunits may mediate native Kir currents responsible for setting the resting membrane potential and might be, at least in part, involved in HCO₃⁻ secretion in pancreatic duct cells. [J Physiol Sci. 2008;58 Suppl:S210]

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In rat submandibular acinar cells, the effects of intracellular Cl⁻ concentration ([Cl⁻]_i) on intracellular Ca²⁺ concentration ([Ca²⁺]_i) were examined. ACh evoked a biphasic increase in [Ca²⁺]_i, that is, an initial transient phase followed by a sustained phase. The initial transient phase is induced by a Ca²⁺ release from the intracellular stores and the sustained phase is maintained by a Ca²⁺ influx from the extracelluar solution, which is the so-called store-operated Ca²⁺ entry. In present study, application of Cl⁻ free solution replaced Cl⁻ by NO₃⁻ increased the sustained [Ca²⁺]_i) level and addition of Gd³⁺ to the Cl⁻ free solution decreased the [Ca²⁺]_i. In the resting state without ACh stimulation, application of Cl⁻ free solution increased [Ca²⁺]_i, suggesting that [Cl⁻]_i decrease activated non-store-operated Ca²⁺ entry. The sustained [Ca²⁺]_i) level increased when Cl⁻ influx was inhibited by an Na+/K+/2 Cl⁻ cotransporter blocker, BMT (bumetanide). These experimental data suggested that [Cl⁻]_i) decrease activated Ca²⁺ entry. When NPPB (5-nitro-2-(3-phenylpropylaminno)-benzoic acid), Cl⁻ channel blocker was applied the sustained [Ca²⁺]_i) level decreased, suggesting that [Cl⁻]_i) increment inhibited Ca²⁺ entry. In conclusion, the present study suggests that [Cl⁻]_i) regulates Ca²⁺ entry pathways which involve store-operated Ca²⁺ channels and non-store-operated Ca²⁺ channels in rat submandibular acinar cells. [J Physiol Sci. 2008;58 Suppl:S210]

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The Na⁺/H⁺ exchanger (NHE) plays a central role in regulating the intracellular pH in cardiac myocytes. We have developed a kinetic model of NHE which could put together major experimental findings. The ion transporting was described as 6-state and 8-state model to embody the process of ion binding and transitions. Proton-regulatory site was also considered with both intracellular and extracellular H⁺ binding sites. The activation by intracellular H⁺ was expressed with a Hill equation with various coefficients, and the effect of extracellular H⁺ was presumed inhibitory. Then, a Levenberg-Marquardt fitting method was applied to the experimental data. Plenty of minima of chi-square function were sought out using numerous starting points. Subsequently, the parameter sets were selected which satisfied the established properties of NHE. Comparison of the least chi-square revealed that the 8-state model with the activation Hill coefficient 3 gave better fitting results. To examine the validity of the model, it was implemented to a simple cell model which was composed of intrinsic pH buffer, Na⁺/K⁺ pump, NH₃/NH₄⁺ flux and others. When the change of intracellular pH was well reproduced during an acid load, the accompanying change of [Na⁺]_i was compatibly simulated. In conclusion, our new NHE model is applicable to further investigation into the pH homeostasis under physiological and pathophysiological conditions. [J Physiol Sci. 2008;58 Suppl:S210]

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Sodium-coupled monocarboxylate transporter 1 (SMCT1, SLC5A8) is expressed in the intestine and kidney. It mediates the transport of short-chain fatty acid, nicotinate and lactate. SMCT1 is suggested to be the important factor that may modify the transport function of URAT1 that transports urate in exchange for intracellular lactate or nicotinate. Since PDZ motif exists at the intracellular C-terminus of SMCT1, it may interact with some PDZ proteins. In this study, to investigate the putative SMCT1-associated proteins, we performed a yeast two-hybrid screen of a human adult kidney cDNA library using the SMCT1 C-terminal tail (SMCT1-CT) as bait. we obtained 22 positive clones. Of these, 13 clones were the multivalent PDZ domain-containing protein PDZK1. Deletion of the SMCT1 C-terminal PDZ motif abolished the interaction with PDZK1. And, the first and third PDZ domains of PDZK1 associate with the SMCT1-CT. Localization of SMCT1 was detected at the apical side of renal proximal tubules. The association of hSMCT1 with PDZK1 enhanced [³H]nicotinate transport activites in HRPE cells (1.7-folds). The elucidation of these interactions may lead to the further understanding of the function and regulation of urate transport via monocarboxylate handling in human kidney. [J Physiol Sci. 2008;58 Suppl:S211]

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Chinese herbs (CH) have been used to relieve xerostomia (dry mouth) by increasing salivary secretion. To examine CH as a new tool to investigate fluid secretion, we have examined 20 CH using rat salivary glands. The submandibular gland of Wistar male rat was surgically isolated and vascularly perfused. The CH concentration in the perfusate was prepared to be equal with the plasma concentration under clinical application. When carbachol (CCh) was given at 0.2 μM as control, saliva secretion formed an initial ephemeral peak at 30 s and followed by gradual increase to a sustained level. Whereas CH alone induced no or little saliva in all kinds of CH chosen. During perfusion with CH, overloading of CCh promoted the fluid secretion in 15 kinds out of 20 kind CH. The promotion was classified to three patterns, which were eventually related to the categories of CH: 1. Overall sustained phase was continuously raised (yin-nourishing agent). 2. The sustained secretion rose to reach maximum then decreased slowly (qi-enhancing agent or heat-clearing agent), 3. Sustained secretion rose to reach the higher maximum then sharply decreased to a lower sustained level (blood activating agent). We picked up 3 CH from each category and measured oxygen consumption (QO₂) and dye secretion, to check energy metabolism and paracellular transport. The QO₂ increased by the 3 chosen CH, but dye secretion was not changed significantly. From the findings the activation of energy supply was suggested as one of possible steps to promote the fluid secretion. [J Physiol Sci. 2008;58 Suppl:S211]

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G-protein coupled signal transduction plays a significant role in nutrient sensing in the gut. Luminal propionate increased transient chloride secretion and Isc in rat colon. This Isc inceases was inhibited by luminal procaine, and serosal atropine and TTX, indicating via a mucosal sensory mechanism. This study aimed to examine an involvement of G-protein signal transduction in Isc response to luminal propionate sensing in rat distal colon. The mucosa-submucosa preparations from the distal colon of rats were used in all experiments. The increase in Isc induced by luminal propionate was inhibited by various types of G-protein inhibitors. This increase evoked by serosal acetylcholin and electrical field stimulation was inhibited by prior addition of luminal GDPβS, and not mastoparan (MP) or methyl-β-cyclodextrin (MβCD). The inhibitory effects of MβCD and MP indicated the involvement of caveolae and Gα_q/11. A 10-min stimulation with luminal propionate (5mM) caused uncoupling of Gα_q/11 and caveolin1 in the cholesterol rich membrane domain. The present results suggest that the secretory response to luminal propionate sensing in the rat distal colon is mediated via G-protein signal transduction. [J Physiol Sci. 2008;58 Suppl:S211]

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We have reported that mast cell surface antigen-1 (MASA-1) is specifically expressed on mast cells in rat type I allergy and mouse atopic dermatitis (NC/Nga mice) models. Additionally, expression of MASA-1 on a human mast cell line, HMC-1, was shown to be up-regulated by TGF-β1 and TNF-α. Herein, we investigated the expression of mouse MASA-1 on mast cells derived from mouse bone marrow cells.Bone marrow cells from BALB/c mice were cultured for 4 weeks in the presence of SCF and IL-3 in order to differentiate bone marrow-derived mast cells (BMMC). Subsequently, cells were co-cultured for 4 more weeks with a primary culture of fibroblasts from the skin of an identical mouse. Mast cells were predominant at 4 weeks of culture. However, the cell number gradually decreased in suspension culture. Co-culture with fibroblasts significantly improved the survival of BMMC.Cultured bone marrow mast cells expressed both FcεRI and c-kit (82.6 ± 1.8 and 90.2 ± 0.2%, at 4 weeks of culture and at 4 weeks of co-culture) by FACS analysis. However, the percentage of MASA-1⁺c-kit⁺ cells was less than 2.5% at 4 weeks of culture. After 4 weeks of co-culture, an average of 11.5% of mast cells were positive for MASA-1. Similar to rat MASA-1, the molecular weight of mouse MASA-1 on immunoblotting was about 90 kDa. MASA-1 protein levels were higher in BMMC co-cultured with fibroblasts than in BMMC cultured without fibroblasts. These results suggest that mouse MASA-1 expression is up-regulated by direct interaction between BMMC and fibroblasts. [J Physiol Sci. 2008;58 Suppl:S211]

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In the last decade, we have reported that sphingosylphosphorylcholine (SPC) induces Rho-kinase-mediated Ca²⁺-sensitization of vascular smooth muscle contraction, thereby leading to vasospasm in vivo. Indeed, SPC concentrations in cerebrospinal fluid are much higher in vasospastic patients after subarachnoid hemorrhage than in non-vasospastic ones. Although coronary vasospasm is also of critical importance in ischemic heart diseases, the serum SPC concentrations were not examined in such vascular diseases. Therefore, as an initial approach to assess clinical importance of SPC in vascular diseases, we aimed to measure the SPC concentrations of the sera obtained from 132 healthy human volunteer donors on regular medical examination, using a high-performance liquid chromatography/tandem mass spectrometry and a deuterated internal control (SPC-d3). The mean and range of serum SPC concentration was 8.02±0.14 nM (mean±s.e.m) and 13.25-2.76 nM, respectively. Furthermore, correlation analysis between SPC concentration and medical examination data supported the independence of SPC on other known parameters with low correlation coefficient of <0.28, although some of them showed statistically significant correlations (P<0.05). [J Physiol Sci. 2008;58 Suppl:S212]

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We study to evaluate the effects of iron-induced oxidative stress and the protective effects of saponin against oxidative damage on rheological properties of erythrocytes. Human erythrocytes were incubated for 1 hour at 37°C with 0-1 mM FeSO₄ in the presence of ascorbate. Thiobarbituric acid reactive substances assay was employed to estimate lipid peroxidation. A cone-plate viscometer, and high-shear rheoscope were used to evaluate the rheological parameters in Fe²⁺/ascorbate-treated erythrocytes. 1) The incubation of erythrocytes with free iron caused a dose-dependent increase in lipid peroxidation, 2) Fe²⁺/ascorbate treatment impaired erythrocyte deformability and erythrocyte suspension viscosity. 3) Saponin partially prevented Fe-ascorbate-induced deformability impairment and decrease viscosity of erythrocyte suspensions. Sapoin is efficacious in protecting erythrocyte's against iron-mediated oxidative injury, which can be attributed to its potent reductant and radical scavenging abilities. [J Physiol Sci. 2008;58 Suppl:S212]

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It is known that the decrease of the blood fluidity is one of the causes in the cardiovascular disorder. The circulatory system is under the control of the autonomic nervous system and endocrine system through the modulation of peripheral vessel resistance and cardiac output. Furthermore blood viscosity changing by the state of platelet aggregation, leukocyte adherence and erythrocyte deformability also affect the blood fluidity. We have reported that blood fluidity shows dynamic changes by the stress, acupuncture, adrenaline receptor agonist and antagonist. In the present study, we examined the influence of heparin-like substance, chondroitin sulfate, in taken orally on the blood fluidity and on the platelet aggregation.We used SPF Wistar male rats, weighing 100g. The experiment group rats were given 20 or 40mg/kg chondroitin sulfate once a day for three days using feeding tube. To the control group, same volume of distilled water was given in same way. The blood sample was mixed with heparin, EDTA-2K or sodium citrate to block their coagulation and testd the fluidity by using Micro Channel Array Flow Analyzer (MC-FAN). Platelet aggregation tendency was examined by the reaction to ADP which activated platelet for aggregation using PA-20(Kowa, Co).In the experimented group, blood fluidity was significantly increased (p< 0.05) and the platelet aggregation formation was also significantly decreased (p< 0.05). The results suggest that chondroitin sulfate increases blood fluidity at least through the prevention of the platelet aggregation. [J Physiol Sci. 2008;58 Suppl:S212]

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We have reported that the low frequency alternative magnetic field stimulation increased blood fluidity.Present study was undertaken to examine the influence of magnetic stimulation on the blood fluidity and on the platelet aggregation which is one of the main factors to change blood fluidity.Specific-pathogen-free Wistar rats weighing 100g were used in the experiment. Animals were divided into two groups. Animals in the control group were put on the apparatus of magnetic stimulator for 30 min without switching on. Animals in the experiment group were stimulated by alternative magnetic field (50Hz, 80mT) for 30 min. Blood samples were collected from the portal vein under the nembutal anesthesia.The sample was mixed with heparin, EDTA-2K or sodium citrate to block their coagulation, and checked the fluidity by using Micro Channel Array Flow Analyzer (MC-FAN).Platelet aggregation tendency was examined by the reaction to ADP which activated platelet for aggregation using PA-20 (Kowa, Co.)As the result, the blood fluidity in the experimented group was significantly increased (p<0.05) and the platelet aggregation formation was also significantly decreased (p<0.05) comparing to that of control group.The results suggest that the magnetic stimulation increased blood fluidity at least through the prevention of the platelet aggregation. [J Physiol Sci. 2008;58 Suppl:S212]

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The habutobin cDNA was cloned from total RNA extracted from venomous glands of Trimeresurus flavoviridis (the habu snake). The conceptual translation of 1539 bp of habutobin cDNA consists of 236 amino acids and its molecular weight is 25.7 kD. Histidine (His)-tagged recombinant habutobin fusion protein, pET-r-habutobin and AcNPV-r-habutobin, was purified by bacterial system and baculoviral system, respectively. After refolding pET-r-habutobin, there were two protein bands at about 32 and 65 kD, indicating that habutobin might be produced as a monomer protein and processed to form two concatenated protein. Purified AcNPV-r-habutobin dose-dependently increased fibrin forming activity and inhibited collagen-induced aggregation of rabbit washed platelets. Thus, AcNPV-r-habutobin produced by baculoviral system is very useful for study on structure-function relationship, which is necessary for developing an antithrombotic drug from habutobin. [J Physiol Sci. 2008;58 Suppl:S213]

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We investigated whether the structure of actin in rat aortic endothelial cells (RAECs) were changed to form the filopodia, lamellipodia and stress fiber on stimulation by bound-thrombin (B-th). In our previous study, we demonstrated that B-th, composed of α-thrombin and fibrin derivatives, passed through the RAECs monolayer. Furthermore, we have shown that B-th cleavaged the thrombin receptor (TR) on the membrane of RAECs, as well as native-thrombin (N-th). The developing mechanism of a paracellular holes, which was formed by B-th, was not clarified. In order to analyse the intracellular signal transduction pathway on stimulation by B-th, we investigated the phosphorylation of actin using mouse monoclonal antibody (SPM101). In addition, we investigated the relationship between actin-cytoskeletal organization and phosphorylation of intracellular protein on stimulation by B-th. Paracellular holes were increased to form with a increase of incubation time of B-th in the RAECs layer. The phosphorylation of actin was also reduced by stimulation of B-th but it was maintained under the presence of Okadaic acid. On immunohistochemical staining, Rho A converged time-dependently after incubated by B-th at the central portion of RAECs. Our findings suggested that RAECs layer permeability is regulated by dephosphorylation of actin and translocation of Rho A. Subsequently, RAECs permeability was increased via the formation of actin stress fiber by the dephosphorylation of actin. [J Physiol Sci. 2008;58 Suppl:S213]

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[Background] After secretion from Weibel-Palade body in vascular endothelial cells (VECs), von Willebrand factor (vWF) circulates as ultra-large multimer forms (ULM-vWF) and adheres to subendothelial collagen, to which platelets subsequently adheres to form platelet thrombi. Lacking of vWF leads to bleeding tendency (vWF disease) and excess amounts of ULM-vWF leads to thrombotic disease (thrombotic thrombocytopenic purpura). [Aim] Employing gene deficiency mouse of vWF cleaving enzyme (ADAMTS13) hybridized with green fluorescent protein (GFP) expressing transgenic mouse (KO mouse), we analyzed the dynamics of vWF exocytosis and platelet adhesion by intra-vital confocal microscopy. [Results] (1) DDAVP, known to stimulate vWF exocytosis, increased ULM-vWF which was visualized as long strings using fluorescent labeled anti-vWF antibody. (2) The strings were longest at 5th minute, and longer in KO mice (5.28±4.28 vs 2.89±2.08 μm). (3) GFP expressing platelets adhered to being secreted ULM-vWF. (4) After topical infusion of 2.5% FeCl2, longer (26.34±8.28 vs 10.73±3.11 μm) and more stable strings were seen in KO mice. [Conclusion] (1) ADAMTS13 appeared to cleave ULM-vWF when it is being secreted. (2) The enhanced secretion itself, however, did not develop microthrombus even in KO mice. (3) Longer and more stable ULM-vWF adhered to injured VECs by ferric chloride treatment in KO mice. [J Physiol Sci. 2008;58 Suppl:S213]

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Magnetic resonance imaging (MRI) is an important modality with no radiation exposure, used daily for clinical diagnosis. MR image is originally developed based on discovery the difference of relaxation time of water protons between tumor and normal tissues.Although MRI has rapidly spread clinically, the origin of the difference has not been resolved. Our recent experiment with nuclear magnetic resonance (NMR) on frog skeletal muscle revealed that tissue water is distinctly classified into 4 groups, whose characteristic relaxation time is T₂ >0.4 sec, T₂ around 0.15 sec, 0.03 sec < T₂ < 0.06 sec, T₂ < 0.03 sec. MRI analysis of muscle tissue of human forearm in vivo, tissue water is classified into two groups which are well consistent with water groups revealed by NMR measurement considering a limitation of data accumulating time in MRI. To elucidate a mechanism of signal intensity in tumor in MRI, further analysis obtained other normal human organs would be needed considering histological and cellular physiological feature for the first step. MRI analysis of skeletal muscle, brain, testis will be shown. [J Physiol Sci. 2008;58 Suppl:S213]

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Serum SH groups can be classified into binding-SH (B-SH), in which SH groups form disulfide bonds with molecules such as cysteine and glutathione, and free-SH (F-SH), in which SH groups are in an unbound state. The sum of these SH groups is referred to as total-SH (T-SH). These groups vary due to hemodialysis and changes in blood oxygen levels. Herein, we induced acute and chronic liver injury by administering carbon tetrachloride and investigated the resulting changes in serum SH groups and liver function.Although hepatogenous enzymes increased with onset of liver injury, the rate of increase differed between acute and chronic liver injury. Regarding serum SH groups, significant increases were observed for F-SH, B-SH, and T-SH in the acute liver injury group. Comparison of increases in F-SH and B-SH showed that increases in B-SH were clearly greater. F-SH was thought to increase based on reactivity, and its binding with molecules causing liver injury was thought to have led to increases in B-SH. Although F-SH increased in the chronic liver injury group, this increase was smaller than that in the acute liver injury group. In addition, B-SH tended to decrease, while T-SH did not change. This finding suggests that long-term liver injury leads to excessive binding of serum SH groups to molecules causing liver injury and production of B-SH in a state that could not be measured using the present method. [J Physiol Sci. 2008;58 Suppl:S214]

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Backgrounds & Aims: The importance of urease-derived ammonia in the pathogenesis of Helicobacter pylori (H. pylori)-induced gastric diseases is known by the fact that Helicobacter felis (expresses urease but not vac A or cag PAI-related gene products) induces gastritis and gastric cancer in animal models. Glutamate is previously reported to protect cultured gastric epithelial cells against ammonia (Nakamura & Hagen, Am J Physiol 283: G1264, 2002). However, whether glutamate protects in vivo gastric mucosa against H. pylori is still unknown. Thus, we examined the effect of glutamate supplementation on H. pylori-induced gastric mucosal damage using gerbil model.Materials & Methods: The diets with or without glutamate supplementation were fed to H. pylori-infected Mongolian gerbils for 3 months, then the gastric mucosa were used for the macroscopic, histochemical analyses. The number of viable H. pylori in the stomach was also determined. Results & Discussion: Glutamate supplementation significantly suppressed H. pylori-induced gastric mucosal damage and inflammatory cell infiltration. Interestingly, the number of viable H. pylori in the stomach was unchanged between the diet with and without glutamate.Conclusion: Glutamate supplementation protects H. pylori-induced gastric mucosal damage without affecting H. pylori itself, suggesting the stimulatory role of glutamate on gastric defensive factors. [J Physiol Sci. 2008;58 Suppl:S214]

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SH-2 containing inositol 5'-phosphatase 2 (SHIP2) is a family of inositol 5'-phophatases, which possess the 5'-phosphatase activity that hydrolyzes PI(3,4,5)P3 to PI(3,4)P2. SHIP2 negatively regulates metabolic signaling of insulin via 5'-phosphatase activity. It was reported that a high fat diet failed to induce obesity in mice lacking the SHIP2 gene and that the enhanced expression of SHIP2 was observed in db/db mice. Moreover, we reported an association of SHIP2 gene polymorphisms (334 C to T) and hyperglycemia (Pancreas 2006). We hypothesized that insulin resistance might be observed in SHIP2 gene transgenic mice. According to a previous report (Genomics 1999), we constructed SHIP2 transgene vector, and was microinjected in C57BL/6 female mice. We succeeded to get three founder mice (1 male and 2 female mice). After SHIP2 transgenic mice have been developed, we examined the changed in body weight and in energy metabolism with or without high fat diet. Glucose tolerance test and insulin test were also examined. Transgenic mice seem healthy and fertile. When mice were maintained with normal diet (CRF-1), either of parameters did not differ between wild-type and transgenic mice. When a high fat diet was provided, blood glucose levels were lower in transgenic mice compared with wild-type mice. Body weight and energy consumption tended to be lower in transgenic mice. In conclusion, SHIP2 transgenic mice unexpectedly revealed resistance to obesity and glucose intolerance when a high fat diet was provided [J Physiol Sci. 2008;58 Suppl:S214]

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We previously reported that mast cell surface antigen-1 (MASA-1) is a specific molecular marker of mast cells. Detection of this protein is thought to be useful in the clinical diagnosis of type I hypersensitivity reactions, and rabbit anti-human MASA-1 (hMASA-1) polyclonal antibody has already been prepared by our laboratory. Therefore, we attempted to prepare mouse anti-hMASA-1 monoclonal antibody. Female BALB/c mice were immunized with recombinant hMASA-1 protein. After several antigen boosters, the spleen was dissected and splenocytes were hybridized with myeloma P3-X63-Ag8.U1. Positive clones were selected by enzyme immunoassay (EIA), and isotypes of secreted antibodies were determined. Bronchoalveolar lavage fluid from patients with lung cancer, sarcoidosis and eosinophilic pneumonia were then studied immunohistochemically (using mast cell tryptase as a common mast cell marker). The antibody appeared to recognize a large number of tryptase-positive mast cells, but there were few MASA-1-positive/tryptase-negative cells. Mast cells were previously recognized using anti-tryptase antibody; thus, the present results suggest that some mast cells are tryptase negative. In order to construct an ELISA system for the high-sensitivity detection of MASA-1, further refinement of assay conditions is necessary; however, the present method represents a promising approach for clinical applications. [J Physiol Sci. 2008;58 Suppl:S214]

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It is hypothesized that the higher population of Ki-67 positive cells at the invasive front of tumor occur the more frequent metastasis in human oral squamous cell carcinomas (OSCCs). To test the hypothesis, we investigated the relationship between the number of tumor cells expressing Ki-67 and the frequency of lymph node metastasis in 44 surgically treated patients with OSCCs. All participants provided informed consent to participate in this study. None of the patients showed evidence of lymph node metastasis at initial clinical evaluation. Immunohistochemical study with anti-Ki-67 monoclonal antibodies was performed to quantitate the number of tumour cells expression Ki-67 in the formalin-fixed, paraffin-embedded biopsy samples and the acquired images were analyzed using NIH image. In 16 of 44 patients, secondary lymph node metastasis occurred. As compared with other Grade types of OSCCs, the frequency secondary lymph node metastasis was increased in Grade 4C and 4D type, in which the number of tumor cells expressing of Ki-67 was increased. These results suggest that the expression Ki-67 in tumor cells may promote secondary lymph node metastasis by lympangioplasty. Subsequently, the release of VEGF from cells expessing Ki-67 in OSCCs may increase the lymphangioplasty. [J Physiol Sci. 2008;58 Suppl:S215]

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[Background] Experimental autoimmune uveoretinitis (EAU) is a cell-mediated disease that is induced in animals sensitive to several retinal antigens. The recruitment of leukocytes is crucial for ocular inflammation in EAU, whereas the target cells and the effector cells have not yet been clearly characterized. [Methods] EAU was induced in B10RIII mice by immunization with human IRBP peptide 161-180 in emulsion with CFA supplemented with M. tuberculosis. Disease severity was assessed clinically by fundoscopic examination. We treated retinas from normal or EAU mice with proteases and prepared retinal monodispersed cells from normal mice as a candidate of target cells and those from EAU mice as a candidate of effector cells. The cytotoxic activity was defined by ⁵¹Cr or LDH release assay. The candidates of target cells were also explored either by opsin RT-PCR experiments or by comparing electron microscopic features of cells from retina of untreated mice with those from retina of EAU mice. [Results] Retinal damage during EAU was initiated by non-phagocytic destruction of inner segments of photoreceptor cells, followed by the phagocytic activity of macrophage against outer segments of photoreceptor cells and fragments of retinal pigmented epithelial cells. [Conclusion] EAU was initiated by non-phagocytic destruction of inner segments of photoreceptor cells. [J Physiol Sci. 2008;58 Suppl:S215]

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We have reported the Moxibustion (Mox) applied to the acupoint MEIMON (GV4) suppressed severity of murine collagen-induced arthritis (CIA). Mox treatment changed lymphocyte subpopulations in the peripheral blood. Increase of CD25⁺CD4⁺ regulatory T cells was particularly noticed. At the same time, spleen volume increase was observed with elevation of clinical arthritis score. Spleen might have essential relation to the pathophysiological stage of CIA. Therefore, to examine the influence of the immune response in the spleen by Mox treatment, we investigated the change of spleen lymphocyte subpopulations by Flow cytometric analysis. Male DBA 1J mice were separated into 4 groups. Group 1, Control: no treatment. Group 2, Control + Mox: treated with Mox by 1mg moxa cone 5 times a day, 3 days a week, for 2 weeks. Group 3, CIA: immunized with bovine type II collagen twice at 3 weeks interval. Group 4, CIA + Mox: immunized and treated with Mox. Clinical symptom was observed until day 35 in CIA, while the symptom was significantly suppressed in CIA + Mox at day 35. At the initial stage (day 28), spleen volume was significantly increased in CIA and CIA + Mox. The percentage of CD3⁺ T cells was significantly decreased in CIA + Mox. However, the percentage of CD25⁺CD4⁺ T cells and the ratio of CD25⁺ in CD4⁺ T cells were significantly increased in CIA + Mox. Together with previous study, present study suggested that suppressive effect of moxibustion involve alteration of T cell line including increase of CD25⁺CD4⁺ regulatory T cells. [J Physiol Sci. 2008;58 Suppl:S215]

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We have reported that moxibustion to MEIMON (GV4) acupoint suppressed severity of collagen-induced arthritis (CIA) in DBA/1J mice. However, the mechanism in the effect of moxibustion is still not clear. Small burn is often observed by moxibustion, therefore we attempted to evaluate if burning stress is involved in the mechanism of moxibustion's effect. To examine that question, we compared the effect of moxibustion in different dose and different acupoint in CIA mice, and also we measured the corticosterone level in those animals. Male DBA/1J mice were separated into six groups. Group 1: CIA (immunized with bovine type II collagen), Group 2: CIA+GV4 (MEIMON, 1.0mg), Group 3: CIA+GV4 (0.5mg), Group 4: CIA+CV12 (CHUKAN, 1.0mg), Group 5: no treatment control, Group 6: GV4 (1.0mg). Moxibustion: applied moxa cone on the acupoint 5 times a day, 3 days a week, for 2 weeks. We evaluated the severity of arthritis by using arthritis score method in all the groups and determined the plasma corticosterone level on day35. Moxibustion to GV4 (1.0mg) significantly suppressed the arthritis score compared to CIA group. CIA+GV4 (0.5mg) and CIA+CV12 (1.0mg) did not show any change. The plasma corticosterone level in CIA was significantly higher than that of control, though the same level in CIA+GV4 (1.0mg). The results suggested that CIA in mice was not influenced by burning, and acupoint-specific and appropriate heat stimulation was needed in moxibustion treatment for the effective inhibition of arthritis severity in CIA mice. [J Physiol Sci. 2008;58 Suppl:S215]

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