We have previously reported that hypotonic shock stimulates Na+ reabsorption by activating a PTK-dependent pathway and that hypotonic shock causes a decrease in cytosolic Cl- concentration ([Cl-]c) through regulatory volume decrease (RVD) in renal epithelial A6 cells. In this report, we found that hypotonic shock increased tyrosine phosphorylation of src kinase at pY416 (an active site of src kinase) in a manner dependent on the RVD-induced decrease in [Cl-]c. We further found that a decreasse in [Cl-]c caused a significant increase in tyrosine phosphorylation of src kinase at pY416 under an isotonic condition without any effect on tyrosine phosphorylation state of src kinase at pY527 (an inactive site of src kinase). Furthermore, pretreatment with PP2 (a specific inhibitor of src kinase) abolished the hypotonic shock-induced stimulation of Na+ rebasorption and alpha-subunit of epithelial Na+ channel (ENaC) mRNA expression. Taken together these results, it is suggested that hypotonic shock stimulates Na+ rebasorption through induction of alpha-ENaC gene expression by activating src kinase through the RVD-dependent decrease in [Cl-]c. Supported by Grants-in-Aids from JSPS (17590191 and 17390057). [J Physiol Sci. 2006;56 Suppl:S115]

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The maxi-anion channel is widely expressed in animal cells. We have recently demonstrated that this channel fulfils a general physiological function as an ATP-conductive gate for cell-to-cell purinergic signaling. However, the regulatory mechanisms of the maxi-anion channel remain poorly understood. We studied the activation mechanism of the maxi-anion channel in inside-out patches excised from mammary C127 cells. The channels activated upon excision in Ca-free solution in the presence of 1 mM Mg2+ and absence of ATP. Increasing both Ca²⁺ and Mg2+ ion concentration led to a dramatic increase in the rate of channel activation. Half-maximal activation occurred at the concentration of 0.0012 mM for Ca²⁺ ions and 2.8 mM for Mg2+ ions. MgATP added to bath (intracellular) solution greatly suppressed the channel activation with half-maximal inhibition at 0.037 mM. A non-hydrolysable analogue of ATP, AMP-PNP, did not suppress the channel activation suggesting that ATP hydrolysis (presumably, the channel phosphorylation) is necessary for the channel inactivation. When all Mg2+ ions were washed out, the free ATP still suppressed the channel, indicating that binding of free ATP can also close the pre-activated maxi-anion channel. Thus, the regulatory control mechanism of the maxi-anion channel involves divalent cation-dependent steps and possibly phosphorylation. [J Physiol Sci. 2006;56 Suppl:S115]

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Recent studies have shown that permeability of some chloride channels to organic anions, such as glutamate and ATP, is involved in cell-to-cell communication mediated by released organic anions. Previous our studies demonstrated that a maxi-anion channel serves as a conductive pathway for ATP release in a mouse mammary cell line (Sabirov et al., 2001), rabbit kidney macula densa cells (Bell et al., 2003) and rat cardiomyocytes (Dutta et al, 2004). In the present study, the possible relation between expression of maxi-anion channel and ATP release was tested in mouse astrocytes in primary culture. In response to hypoxia stress, astrocytes exhibited both activation of maxi-anion channel and massive release of ATP. Hypoxia-induced ATP release was inhibited by blockers of maxi-anion channel, but not by those of other candidate pathways for ATP release, such as gap junction hemi-channel, CFTR channel, exocytosis and volume-sensitive outwardly rectifying (VSOR) anion channel. Using a biosensor technique based on ATP responses of P2X2 receptors expressed in HEK293 cells, the local ATP concentration on a single astrocyte surface was found to increase to about 5 μM during hypoxia. Therefore, it is concluded that the maxi-anion channel serves as a major pathway for ATP release from astrocytes under hypoxia. [J Physiol Sci. 2006;56 Suppl:S115]

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The platinum-based drug cisplatin is a widely used anticancer drug which acts by causing the induction of apoptosis. Some types of cancer have intrinsic or acquired resistance to cisplatin, however. A model of cisplatin resistance is provided by the cisplatin-resistant KB/CP4 human epidermoid cancer cell line. It was found previously in our laboratory that activity of the volume-sensitive, outwardly rectifying chloride channel (VSOR-ClC) is virtually absent in KB/CP4 cells. We hypothesized that the lack of VSOR-ClC current may contribute to cisplatin resistance in these cells. An attempt was made to restore the current in KB/CP4 cells so that the effect of its expression on cisplatin resistance could be tested. Treatment of KB/CP4 cells with trichostatin A (TSA), a histone deacetylase inhibitor, caused VSOR-ClC current to be partially restored. A cell viability assay showed that in response to cisplatin, viability of cells treated with TSA for 48 h decreased significantly compared to control cells. Moreover, a caspase-3 activity assay showed that TSA-treated cells underwent significantly increased apoptosis induced by cisplatin. These effects were blocked by simultaneous treatment of the cells with a VSOR-ClC blocker. From these results, we conclude that restoration of VSOR-ClC functional expression by TSA treatment leads to a decrease in cisplatin resistance and an increase in cisplatin-induced apoptosis in KB/CP4 cells. [J Physiol Sci. 2006;56 Suppl:S115]

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It has recently been reported that extracellular acid pH activates anion channels in several cell types. In the present study, we also found functional expression of proton-activated Cl ⁻ channel in human epithelial HeLa cells. Whole-cell currents were rapidly activated by extracellular acidic solution (pH < 5.0) in a reversible manner. This current exhibited prominent outwardly rectification under symmetrical Cl ⁻ conditions, time-dependent activation at positive potentials, and low-field anion permeability sequence of I ⁻ > Br ⁻ > Cl ⁻ > aspartate ⁻. A Cl ⁻ channel blocker, 4, 4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) or phloretin, could inhibit the current in a concentration-dependent manner with the IC₅₀ value of 0.12 or 18.6 μM, respectively. Anion selectivity and sensitivity to Cl ⁻ channel blockers of this proton-activated current are similar to those of the volume-sensitive outwardly rectifying (VSOR) Cl ⁻ channel current in HeLa cells. However, the other properties are distinct from those of the VSOR Cl ⁻ channel which is inhibited by acid and exhibits modest outward rectification and inactivation kinetics at positive potentials. [J Physiol Sci. 2006;56 Suppl:S116]

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Whereas the Ca²⁺-dependent contraction of vascular smooth muscle (VSM) which regulates physiological vascular tone, the Rho-kinase (ROK)-mediated Ca²⁺-sensitization of VSM contraction contributes to abnormal VSM contraction such as vasospasm. We previously found that sphingosylphosphorylcholine (SPC) is an upstream messenger for the ROK-mediated Ca²⁺ sensitization and that inhibitors of Src family tyrosine kinase (Src-TK) blocked the SPC-induced contraction and activation of ROK. In the present study, we attempted to determine the enzyme molecule in a family of Src-TK which contributes to the Ca²⁺-sensitization mediated by a SPC/ROK pathway. In order to accomplish this purpose, we performed knockdown of the target molecule by using siRNA which was transfected into the human coronary artery smooth muscle cells (CASMCs) with the efficiency of about 100%. The siRNA-mediated knockdown of Fyn inhibited the SPC-induced contraction of CASMC, whereas non-silencing control siRNA lacked any effect. These results provide the first direct evidence that Fyn mediates the Ca²⁺-sensitization of VSM contraction induced by a SPC/ROK pathway. In addition, Fyn constructs (wild, constitutively active, and dominant negative types) were transfected to CASMCs with high efficiency (> 50%), although CASMCs were well-differentiated contractile cells. In poster presentation, the effects of transient overexpression of Fyn constructs on the contraction of CASMCs will be also discussed. [J Physiol Sci. 2006;56 Suppl:S116]

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Phospholipase C-zeta (PLCζ) is a strong candidate of the mammalian sperm factor that induces IP₃-mediated Ca²⁺ oscillations and subsequent embryonic development. PLCζ consists of 4 EF-hand domains (EF1-4) in the N terminus, X and Y catalytic domains, and C2 domain in the C terminus. PLCζ expressed by injection of cRNA into mouse eggs causes fertilization-like Ca²⁺ oscillations, and then it is accumulated into the formed pronucleus as the sperm factor is. The nuclear translocation ability (NTA) was investigated by expressing PLCζ mutants tagged with a fluorescent protein by RNA injection into eggs or 1-cell embryos. Point mutation analysis revealed a lysine-rich nuclear localization signal (NLS) sequence between Lys³⁷⁴ and Lys³⁸¹ in the X-Y linker region. Truncation of EF1 resulted in the loss of NTA, and point mutation revealed a responsible sequence in the N terminus of EF1. However, even if EF1 was present, NTA was lost when EF2-4 or C2 domain was deleted. Both NTA and Ca²⁺ oscillation-inducing ability are lost in these truncation or deletion mutants. Similar results were obtained in cultured COS cells after transfection with cDNA of mutants. It is predicted from the 3-D structure of PLCδ1 that PLCζ is folded at the hinge region in the X-Y linker and that EF-hand domains and C2 domain make extensive contact. Besides NLS, highly coordinated overall structure of PLCζ is responsible for NTA as well as Ca²⁺ oscillation-inducing activity. [J Physiol Sci. 2006;56 Suppl:S116]

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PACAP and VIP are closely related neuropeptides and act primarily as inhibitory transmitters on most of the gastrointestinal and vascular smooth muscle cells. However, their actions are opposite in the guinea pig gallbladder. In this study we have tried to identify PACAP/VIP receptor subtypes expressed in the guinea pig gallbladder. Total RNA was extracted from the guinea pig gallbladder. RT-PCR was conducted using the primers with high sequence homology among human, mouse, and rat PAC1, VPAC1, VPAC2 receptors. PAC1, VPAC1 and VPAC2 receptor mRNAs were abundantly expressed in the gallbladder. Sequence analysis of guinea pig PAC1 receptor revealed a high homology (91% in nucleotide sequence and 99% in amino acid) to human PAC1 receptor. There was an isoform of PAC1 receptor that contained an additional 84 nucleotides encoding 28 amino acids in the third intracellular loop. The amino acid sequence was identical to that of the hop variant reported in rats and humans. The nucleotide and amino acid sequences of guinea pig VPAC1 and VPAC2 receptors also had high homologies to the respective human (90% and 95%), rat (91% and 93%), and mouse (93% and 91%) sequences. The guinea pig gallbladder express PAC1, hop variant, VPAC1 and VPAC2 receptor mRNAs. The expression of the hop variant of PAC1 receptor may be related to the contractile response observed in the gallbladder. [J Physiol Sci. 2006;56 Suppl:S116]

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Signal mechanisms of regulatory volume increase (RVI) in HeLa cells and of RVI inhibition under apoptotic stimulationTakahashi, Nobuyuki; Subramanian, Muthangi; Okada, Yasunobu (Dept. Cell Physiol., Natl. Inst. Physiol. Sci., Okazaki, Japan)Most cells show cell volume recovery, called regulatory volume increase (RVI), after osmotic shrinkage. However, under apoptotic conditions, cell volume persistently decreases without exhibiting RVI. In human epithelial HeLa cells exposed to hypertonic solution, RVI was significantly inhibited by an Akt blocker. Moreover, exogenous expression of the dominant negative form of Akt inhibited RVI under hypertonic conditions. Akt was phosphorylated by hypertonicity, and this phosphorylation was inhibited by apoptotic stimulation by staurosporine, H₂O₂, or TNF-α. Either of these apoptotic stimuli suppressed RVI and then induced apoptotic cell death. Apoptosis signal-regulating kinase 1 (ASK1) was found to be activated by either apoptosis inducer. Overexpression of the kinase dead mutant of ASK1 restored both shrinkage-induced Akt phosphorylation and RVI under apoptotic conditions. Thus, it is concluded that Akt activation induced by hypertonicity is involved in the RVI mechanism in HeLa cells and that shrinkage-induced Akt activation is inhibited by ASK1 activated by various apoptotic stimuli thereby leading to persistent cell shrinkage in apoptotic cells. [J Physiol Sci. 2006;56 Suppl:S117]

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NK (Natural killer) cells have the ability to migrate and eliminate tumor cells. We evaluated the role of matrix metalloproteinases-1 (MMP-1) on regulating chemokine-dependent invasion of NK cells into type I collagen. We revealed that CXCL12 promoted the invasion program on freshly isolated human NK cells in a MAP kinase dependent manner, because CXCL12 enhanced NK cells invasion was significantly inhibited by p38MAP inhibitor SB203580 and MEK 1/2 inhibitor U0126. Next we examind whether CXCL12 especifically enhanced the production of MMP-1 from NK cells. This production wes significantly inhibited by SB203580 and U0126. Immunofluorescence confocal microscopic studies suggested that MMP-1 was co-localized with alpha2 integrin on the NK cell surface stimulated by CXCL12. The co-localization of MMP-1 and alpha2 integrin was significantly inhibited by SB203580 and U0126. Immunoprecipitation assy showd that productional MMP-1 associated with alpha2 beta1 integrin on NK cells stimulated by CXCL12. This association was significantly inhibited by SB203580 and U0126. These results suggested that MMP-1 associated with the cell surface was involved in NK cell invasion into type I collagen, and MMP-1 associated with alpha2 integrin on the cell surface may be a critical step in facilitating pericellular matrix degradation during cell invasion. [J Physiol Sci. 2006;56 Suppl:S117]

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Hypercholesterolemia is a major risk factor of cardiovascular events. A Rho-kinase-mediated Ca²⁺ sensitization of vascular smooth muscle (VSM) plays a critical role in abnormal vascular contraction such as vasospasm. We found that sphingosylphosphorylcholine (SPC) sequentially activated Fyn and Rho-kinase to induce the Ca²⁺ sensitization. We observed the strong link between the SPC-induced contraction and the tissue and cellular cholesterol in VSM, suggesting the involvement of the cholesterol-enriched membrane microdomains, membrane lipid rafts. In membrane-permeabilized VSM, SPC induced contraction in the absence of cytosolic GTP which is required for the activation of G-proteins and thus of GPCRs. Taken together with the localization of Fyn in the membrane lipid rafts, these findings suggest the importance of cholesterol and are compatible with the interaction of SPC with the other membrane components than GPCRs and/or the direct interaction between SPC and lipid membrane, which may in turn affects the function of membrane proteins. Therefore, we examined the interaction of SPC with raft model membranes. The surface plasmon resonance measurement (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. We propose that cholesterol and its enriched membrane lipid rafts may play a role in Ca²⁺ sensitization mediated by a SPC-Fyn-Rho kinase pathway. [J Physiol Sci. 2006;56 Suppl:S117]

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Glutamate may cause Ca²⁺ entry through Ca²⁺-permeable glutamate receptors, which in turn stimulates the anti-apoptotic signaling cascade in glioma cells. Here, we found that a human glioma cell line, U-87 MG, expressed GluR1, GluR2 and GluR3 subunits of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate acid-type glutamate receptors (AMPARs). Approximately 20% of GluR2 was expressed in the unedited form, which is known to assemble Ca²⁺-permeable AMPARs. Ca²⁺ entry through the activation of these receptors by AMPA was detected clearly in approximately 20% of U-87 MG glioma cells. Cell proliferation assays revealed that the application of AMPA or glutamate facilitated cell proliferation by activating AMPARs in low-serum medium containing 0.5% fetal calf serum (FCS). Unexpectedly, cell proliferation by the activation of AMPARs was not detected in serum-rich medium containing 10% FCS. Overexpression of the unedited form of GluR2 (GluR2Q) by adenoviral-mediated gene transfer markedly increased the Ca²⁺ entry into U-87 MG cells. This treatment in the presence of glutamate facilitated proliferation and migration of U-87 MG cells in the low-serum condition, whereas it had again no effect in the serum-rich condition. It is therefore likely that cell proliferation and migration of U-87 MG cells are under the regulation of growth factors contained in the serum as well as Ca²⁺ entry through AMPARs, and that the latter regulation becomes evident only when serum factors are deprived of culture medium. [J Physiol Sci. 2006;56 Suppl:S117]

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Although cell shrinkage is one of the phenotypical features of apoptosis, it has been controversial whether it is a prerequisite to apoptosis induction. In this study, we examined whether a persistent decrease in cell volume could per se initiate apoptotic cell death without any apoptotic stimulus. When HeLa cells were incubated in isotonic Na⁺-free solution, the mean cell volume immediately began to decrease and reached 84% of the original value within 30 min. After persistent shrinkage, activation of caspase-3 and reduction of cell viability were observed. Application of a blocker of the Na⁺-K⁺-2Cl⁻ cotransporter (NKCC), bumetanide (100 μM) or furosemide (1 mM), or that of the Na⁺/Ca²⁺ exchanger (NCX), KB-R7943 (25 μM), inhibited Na⁺ deprivation-induced shrinkage and attenuated apoptotic cell death. These results suggest that shrinkage of HeLa cells exposed to Na⁺-free solution is induced by efflux of Na⁺, K⁺ and Cl⁻ mediated by operation of NKCC and reversed operation of NCX, and that an isotonic volume decrease per se triggers the apoptotic process. [J Physiol Sci. 2006;56 Suppl:S118]

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Sphingosine-1-phosphate (S1P) receptor is known to show a variety of actions including endothelial permeability regulation. Multiple S1P receptors including S1P-1 are expressed on pancreatic islets, and S1P, a S1P-1 receptor agonist, was shown to potentiate insulin secretion. However, a precise mechanism of the receptor action is not clear at moment. Using Cre-LoxP system, we made mice specifically lacking S1P-1 receptor gene in pancreatic islet B-cells. The blood glucose levels in fasting state are the same in knockout and control mice. After intraperitoneal glucose challenge, knockout mice were significantly less able to normalize blood glucose levels than were the control mice. In the perifusion experiment for isolated islets, glucose stimulation increased insulin secretion in the control islets, whereas it failed in islets from knockout mice. In isolated pancreatic B-cells from both control and knockout mice, glucose stimulation caused depolarization followed by action potential firing. The membrane capacitance measurement revealed that calcium pulse stimulation could not increase the exocytosis of insulin granules. These results indicate that S1P-1 receptors are essential for B-cell insulin secretion in the exocytotic process at least distal to calcium signals. [J Physiol Sci. 2006;56 Suppl:S118]

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Phosphatidylinositol-4,5-bisphosphate (PIP₂) is well known to be a source of the important second messengers inositol-triphosphate (IP₃) and diacylglycerol (DAG). To investigate the intracellular localization of PIP₂, it has been commonly utilized pleckstrin homology (PH) domain fused with green fluorescent protein (PH-GFP) as a marker. However this method is not suitable for cells difficult to be transfected. In the present study, we established the assay method of receptor-induced transient dissociation of PIP₂ from the plasma membrane in rat brown adipocytes using anti-PIP₂ antibody. Cells incubated with or without stimuli for 0-120 sec were fixed immediately, blocked with BSA and incubated with anti-PIP₂ antibody. After washing, the cells were incubated with Alexa Fluor 546-labeled IgG and fluorescent signals were observed using confocal laser scanning microscope. In control cells, PIP₂ displayed staining which outlined the cells periphery. When the cells were stimulated with 1μM noradrenaline (NA), plasma membrane PIP₂ was rapidly decreased within 2.5 sec. Dissociation of PIP₂ from the plasma membrane was transient and relocalized to the plasma membrane within 2 min. Stimulation of the cells with 50μM ATP showed similar response to NA. 5μM wortmannin inhibited relocalization of PIP₂ to the plasma membrane in NA- or ATP-stimulated cells, indicating that phosphatidylinositol-4-kinase (PI4K) plays an important role in PIP₂ reproduction. [J Physiol Sci. 2006;56 Suppl:S118]

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Contributions of COX-1 and COX-2 in basal and ACh-stimulated prostaglandin E₂ (PGE₂) release were studied in antral mucosa of guinea pig. PGE₂ was released from antral mucosa spontaneously. Acetylcholine (ACh), which increases [Ca²⁺] _i, increased the PGE₂ release from antral mucosa in a dose dependent manner. COX-1 or COX-2, which generates PGE₂ from arachidonic acid (AA), was regulated by [Ca²⁺]_i. Basal and ACh-stimulated PGE₂ release were increased by the addition of AA, and was inhibited by a PLA₂ inhibitor and COX inhibitors. SC560 (100 nM, a selective inhibitor of COX-1) decreased ACh-stimulated PGE₂ release without any decrease in basal PGE₂ release, while ionomycin increased PGE₂ release. NS398 (20 μM, a selective inhibitor of COX-2) decreased basal PGE₂ release without any decrease in ACh-stimulated PGE₂ release. Moreover, in isolated antral epithelial cells, SC560 inhibited ACh-stimulated-PGE₂ releases, however, NS398 did not. Thus, in antral mucosa, basal PGE₂ release is maintained via COX-2 of interstitial cells and ACh-stimulated PGE₂ release is maintained via COX-1 of antral epithelial cells. These observations suggest that PGE₂ released via COX-2 in the interstitial cells maintains an integrity of the resting antral mucosa and that released via COX-1 in antral epithelial cells maintains an autocrine mechanism, which enhances Ca²⁺-regulated exocytosis in ACh-stimulated antral mucosa, such as during meals. [J Physiol Sci. 2006;56 Suppl:S118]

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Phthalate esters have been used as plasticizers of synthetic polymers. Recent studies revealed that they induce atrophy of the testis, although its pathogenesis remains unknown. Testicular atrophy with aspermatogenesis was induced by feeding with 2% DEHP-containing diet. The biochemical and immunohistochemical analysis revealed that DEHP increased the reactive oxygen species generation, with concomitant decrease of glutathione and ascorbic acid, and selectively induced apoptosis of spermatocytes, thereby causing atrophy. Oxidative stress was selectively induced in germ cells, but not in Sertoli cells, treated with mono(2-ethylhexyl)phthalate (MEHP), a hydrolysed metabolite of DEHP. Furthermore, MEHP selectively induced the release of cytochrome c from mitochondria of the testis. These results indicate that oxidative stress elicited by MEHP principally injured mitochondrial function, and induced apoptosis of spermatocytes and atrophy of the testis. Using the 2% DEHP-dose, the effect of simultaneous administration of vitamins C and E was examined. The vitamin supplementation significantly prevented the testicular injury. Results suggest that antioxidant vitamins can protect the testes from DEHP-toxicity. Some of rare sugars (i.e. D-psicose and D-allose) are also effective in prevention of the testicular injury. Microarray analysis has been applied to elucidate the genes involved in the DEHP-toxicity and the protection mechanism. [J Physiol Sci. 2006;56 Suppl:S119]

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PI3Ks produce 3'-phosphoinositides (3'-PIs) including PI(3,4,5)P₃ and PI(3,4)P₂, whereas PTEN dephosphorylates 3'-PIs to decrease the contents of PI(3,4,5)P₃ and PI(3,4)P₂. Elevation of PI(3,4,5)P₃ and PI(3,4)P₂ contents induces activation of PDK1 and Akt, resulting in cell migration and cell survival. Very recently, it has been shown that Rho-ROCK stimulates PTEN, resulting in inhibition of Akt and cell migration. These observations led us to investigate the effects of Rho-stimulating GPCR agonist LPA on Akt activation and cell migration. In CHO cells that endogenously express LPA₁ receptor, IGF-I stimulated Akt phosphorylation (P-Akt) and chemotaxis in a PI3K inhibitor-sensitive manner. In PTX-treaed cells, LPA inhibited IGF-I-induced P-Akt and chemotaxis. Y-27632, a ROCK inhibitor, prevented LPA-inhibition of IGF-I-induced P-Akt, indicating the ROCK mediates inhibition of P-Akt. However, Y-27632 failed to abrogate LPA-inhibition of IGF-I-induced chemotaxis. Thus, there was a discrepancy between LPA-induced inhibition of the cellular 3'-PIs amount, which is reflected by the extent of P-Akt, and inhibition of cell migration. These results suggest that LPA-induced inhibition of cell migration is not dependent on ROCK-mediated stimulation of PTEN and, thereby, reductions of cellular 3'-PIs contents [J Physiol Sci. 2006;56 Suppl:S119]

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We previously reported that intrathecal administration of various local anesthetics resulted in the dorsal root axonal degeneration near entry into the spinal cord in rats. The level of neurotoxicity of local anesthetics in the dorsal root depended on the kind of anesthetics and this was comparable to the level of side effects occurred in the clinical use. In the present study, we assessed the effects of various local anesthetics on axonal transport in cultured mouse dorsal root ganglion neurons. Lidocaine, bupivacaine, and ropivacaine (concentrations: 1-50 mM) all decreased axonal transport, but in different degree. The order of potency was lidocaine<bupivacaine<ropivacaine. At a concentration of 50 mM, lidocaine and bupivacaine but not ropivacaine caused the rupture of plasma membrane in some dorsal root ganglion neurons. Considering with the pharmacological potency, lidocaine:bupivacaine:ropivacaine = 1:4:4, the neurotoxocity of ropivacaine is likely much less than that of lidocaine. These in vitro experiments may be useful for determining the neurotoxicity of local anesthetics. [J Physiol Sci. 2006;56 Suppl:S119]

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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 Lineage Kinase (MLK) 3. Here we analyzed activity of MLK3 in hippocalcin deficient mice. Immunoblot analysis using substrate specific anti-phospho-antibodies 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. By adding recombinant hippocalcin (100ng) to the assay condition, the activity of MLK3, which was immunoprecipitated from wild type mice, was inhibited. These results indicate that the binding of hippocalcin to C-terminal domain of MLK3 directly inhibits MLK3 kinase activity. [J Physiol Sci. 2006;56 Suppl:S119]

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S1P has a critical role in vascular maturation during mammalian development. However, little is known about the role of S1P in ischemia-induced angiogenesis in adults. We investigated the effect of both exo- and endogenous S1P on angiogenesis in ischemic skeletal muscle in adult mice. Unilateral hindlimb ischemic model is a well-established in vivo angiogenesis assay system. We monitored post-ischemic angiogenesis by blood flow recovery with laser doppler imager and capillary density with anti-CD31 immunohistochemistry after surgery. First, we injected S1P into ischemic muscle everyday after surgery in C57BL6/J mice. Limb blood flow was 2.5 fold elevated in S1P(10⁻⁸M)-injected mice at day 7. Capillary density was 1.5-fold increased in S1P group at day 10. These effects were comparable to bFGF administration. Trying to create long acting S1P, we prepared the slow-release microsphere containing S1P with polylactide-co-glycotide and intramuscularly administrated it just once immediately after surgery. Mice received microsphere containing S1P increased blood flow in ischemic limb. We also examined the effect of endogenous S1P overproduction by generating sphingosine kinase 1 transgenic (SphK1-Tg) mice, and blood flow was slightly increased compared with littermate wild type mice. S1P₁ receptor selective agonist SEW2871 also accelerated blood flow recovery. We showed S1P accelerates ischemia-induced angiogenesis, most likely via S1P₁ receptor in adult mice. S1P and SEW2871 is a potential therapeutic for ischemic diseases. [J Physiol Sci. 2006;56 Suppl:S120]

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1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is commonly used for adjuvant chemotherapy to treat malignant glioma including glioblastoma multiforme (GBM). BCNU kills tumor cells via multiple actions including carbamoylation and alkylation. Herein we test the effects of NO donors on alkylating cytotoxicity to rat C6 glioma cells. The alkylating agents tested included methyl methanesulfonate (MMS), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU). The synergistic effects of three NO donors, namely S-nitrosoglutathione (GSNO), diethylamine NONOate (DEA/NO), and sodium nitroprusside (SNP) on alkylating agents were determined by colony-formation assay. We found that inclusion of NO donors substantially reduced the extents of colony formation following exposure of glioma cells to all three alkylating agents. Among the three NO donors, GSNO appeared to be the most potent one. GSNO also exerted similar synergistic actions reducing the extents of colony formation when co-administrated with 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-hydrazine, another alkylating agent that mimics the chloroethylating action of BCNU. O6-Methylguanine methyl-DNA transferase (MGMT) is a DNA repair enzyme that removes the cytotoxic O6-alkylguanine adducts induced by alkylating agents. Western analysis indicated that expression of MGMT was reduced in the presence of GSNO, suggesting the possibility that GSNO enhanced alkylating cytotoxicity via, at least in part, decreasing cellular MGMT contents. [J Physiol Sci. 2006;56 Suppl:S120]

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Neurotrophins are growth factors that exert multiple actions on neuronal and nonneuronal cells. We have previously shown that neurotrophins, NT-3 and NT-4/5, are expressed in mouse alveolar macrophages. In the present study, we further attempted to clarify the intracellular localization of NT-3 and its co-localization with interleukin IL-1, fibroblast growth factor FGF, and histamine in alveolar macrophages. Immunocytochemical staining with antibodies against NT-3, lysosomal membrane protein LAMP-2, IL-1, FGF, and histamine was performed on alveolar and peritoneal macrophages obtained from the adult mouse. Approximately 80% of alveolar macrophages were immunoreactive for NT-3. In contrast, peritoneal macrophages were rarely immunoreactive for NT-3 (<5%). In these NT-3-immunoreactive macrophages, NT-3 was localized in intracellular granules. The granule localization of NT-3 was confirmed, since the NT-3-containing granules were immunoreactive for LAMP-2, which labels intracellular granule membrane. Almost all alveolar and peritoneal macrophages were immunoreactive for IL-1, FGF, and histamine. IL-1 and FGF were diffusely distributed in the cytoplasm. Histamine was localized in granules. In some granules, histamine was co-localized with NT-3. Since a large number of alveolar macrophages contain NT-3 in their granules and NT-3 is co-localized with histamine in some granules, they may be co-released in an exocytotic manner from alveolar macrophages and may play synergic roles in pulmonary pathophysiology. [J Physiol Sci. 2006;56 Suppl:S120]

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Rat primary microglia (MG) acquired a multipotent property to give rise to neuroectodermal cells through two-step culture in 10 and 70% serum-supplemented media for 5 d (Yokoyama et al., Glia 2004; 45, 96-104). Such multipotent MG called promicroglioblasts (ProMGB) formed cell aggregates, which generated cells with neuroectodermal phenotypes shortly after transfer into serum-free medium. As revealed by immunohistochemistry, there were a few MG expressing NG2-chondroitin sulfate proteoglycan (NG2) in the neonatal rat brain. Primary culture from the neonatal brain contained NG2+ MG, which appeared being the source of NG2+-ProMGB aggregates. The aggregates were MG-markers+/GFAP+/NCAM+/S-100b- and possessed an alkaline phosphatase activity. Marked accumulation of NG2+ MG was observed in the close vicinity of stab wounds made in mature rat brain. The NG2+ MG in the wounds separated with trypsin-EDTA formed NG2+ aggregates in 70% serum-supplemented medium and then turned into cells with neuroectodermal phenotypes in serum-free medium. Although it is quite difficult to separate viable neurons from mature brains, cells from the stab wounds generated process bearing b-tubulin III+ cells easily. These data suggest that NG2+ MG in normal developing or pathologic brains are involved in genesis or regeneration of the brain. [J Physiol Sci. 2006;56 Suppl:S120]

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Although microglial cells are currently considered mesodermal cells, it has not been completely determined whether they are hematopoietic or mesenchymal origins. To obtain some insights in this issue, we induced dedifferentiation of microglial cells in culture by incubating in 70% serum-supplemented medium for 2 d. Microglial cells were separated from primary mixed glial culture that was started from neonatal rat forebrains or from ischemic brain lesions of rats whose right middle cerebral artery was transiently occluded. Cells cultured in 70% serum-medium gradually exhibited amoeboid shape and often formed cell aggregates, while increasing expression of Id genes and getting highly proliferative. Such cell aggregates differentiated into cells with neuroectodermal phenotypes after they were transferred into serum-free medium on poly-L-lysine-coated substrate. By contrast, cells that had been cultured in 70% serum-medium gradually fused resulting in formation of multinuclear giant cells, after they were transferred into 10% fetal calf serum-supplemented medium containing M-CSF and RANKL. As revealed by RT-PCR, such giant cells elevated expressions of mRNAs encoding DC-stamp, TRAP and Cathepsin K that are specific markers of osteoclasts. Taken that osteoclasts are derived from hematopoietic stem cells (HSCs) and HSCs are known to generate neuroectodermal cells, microglial cells may be of hematopoietic origin. [J Physiol Sci. 2006;56 Suppl:S121]

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We found that amoeboid-shaped microglia expressing NG2 proteoglycan accumulated in stab wounds in the brain. Some of the NG2-positive microglia expressing nestin and GFAP turned into cells with neuroectodermal phenotypes in vitro. To elucidate whether such amoeboid NG2-positive microglia are blood-borne or the activated form of resident microglia, we compared the nature of cells expressing microglia markers using three kinds of brain pathology models using Wistar rats; stab-wound, middle cerebral artery occlusion (MCAO), and facial nerve axotomy models. The former two models accompanies breakdown of blood brain barrier (BBB), while the axotomy model does not. A huge number of NG2-positive amoeboid shaped cells expressing Iba1, a marker of microglia/macrophages, accumulated in the stab wounds and the core lesions of MCAO. The majority of the amoeboid cells were proliferating as revealed by Ki67-immunostaining. In contrast, microglial cells in the axotomied facial nerve nucleus enlarged somata but still kept ramified shapes, and none of them were Ki67-negative. Most of resident microglial cells died within 2 days after the stab-lesioning or MCAO, while none of microglia died in the facial nerve nucleus. These observations suggest that multipotent NG2-positive microglia in the brain lesions are blood-borne and distinct from resident ramified microglial cells. [J Physiol Sci. 2006;56 Suppl:S121]

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The anaerobic bacterium Megasphaera elsdenii uses lactate as the carbon source and produces lower fatty acids in the rumen of cattle and sheep. Electron-transferring flavoprotein (ETF) is a key enzyme in the intracellular redox system of M. elsdenii. ETF receives electrons from flavoprotein D-lactate dehydrogenase (D-LDH), which oxidizes D-lactate to pyruvate. The received electrons are then transferred to flavoprotein enoyl-CoA reductase (ECR), which reduces enoyl-CoA to acyl-CoA. The acyl-CoAs are eventually changed to fatty acids by CoA elimination. ETF also receives electrons from NADH, which is reduced by many other redox reactions. ETF contains two FAD molecules as the cofactor. The functions of the two FAD molecules are presently unlcear. In this study, we found the followings by spectrophotometric experiments using purified flavoproteins. Here the two FAD molecules in ETF are designated FAD-1 and FAD-2. (1) NADH reduces both FAD-1 and FAD-2. (2) D-LDH interacts with only FAD-1. (3) ECR interacts with both FAD-1 and FAD-2. (4) Electron transfer between FAD-1 and FAD-2 can occur without NADH/NAD⁺, D-LDH, and ECR. [J Physiol Sci. 2006;56 Suppl:S121]

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[Background] Previously, we found that allograft-induced macrophages (AIM; H-2D^bK^b) were the major effector cells responsible for allograft (e.g., BALB/c skin and Meth A tumor; H-2D^dK^d) rejection. In the last meeting, we reported isolation of a cDNA, which encoded a novel receptor on AIM for allogeneic MHC (H-2D^d), by using anti-AIM monoclonal antibody (mAb; R15) and H-2D^d tetramer. We named this receptor "macrophage MHC receptor (MMR)". In the present study, we obtained a cDNA encoding a novel receptor on AIM for allogeneic MHC (H-2K^d). [Method] cDNA fragments were isolated by the T7 phage expression cloning method using R12 mAb and H-2K^d tetramer. Full length of the cDNA was obtained by the RACE method. mRNA expression was estimated by RT-PCR. cDNAs fused to GFP cDNA were transfected to HEK293T cells; and the binding of H-2 molecules to the transfectants was explored under a confocal microscope. The dissociation constant (K_d) of AIM toward H-2 molecules was assessed by flow cytometry. [Results] We isolated the full length (≈2.4kb) of cDNA, which encoded a receptor on AIM for allogeneic MHC (H-2K^d), and named the receptor 'MMR2'. The MMR2 mRNA was expressed exclusively in AIM but not in other cells infiltrating into allograft. HEK293 cells transfected with MMR2 cDNA reacted with H-2K^d, but not with other H-2, molecules. The H-2K^d binding was completely suppressed by R12 or anti-H-2K^d mAb. The K_d value of AIM toward H-2K^d was 2.8×10⁻⁹ M. [J Physiol Sci. 2006;56 Suppl:S121]

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Tight junction (TJ) is an important adhesion system in epithelial cells, and also plays a role for regulation of paracellular flux across epithelial sheets. Claudins, a component of TJ, comprise a large family consisting of more than 20 members in mammals, and contribute to regulation of barrier function. We have compared expression of claudins in culture and intact duct cells of the rat submandibular gland with that in MDCK cells, a typical epithelial cell line. SMIE cells derived from rat submandibular duct were kindly provided by Dr. B. J. Baum (NIDCR). Duct cells were isolated from rat submandibular gland. TJ proteins were detected by western blotting. Immunofluorecence strain of TJ proteins was observed by a confocal laser scanning microscope. SMIE cells formed TJ as well as MDCK cells, which was confirmed by immunofluorecence strain of the TJ proteins occludin and ZO-1. Claudin-3 protein was detected in both cells. Claudin-1 and claudin-4 proteins were detected in MDCK cells, but not in SMIE cells, which was confirmed by immunofluorecence strain of claudins. However, in rat submandibular duct cells, claudin-4 was detected by western blotting and immunofluorecence strain. In SMIE cells, the transepithelial electrical resistance was lower and the flux of FITC-dextran was higher than in MDCK cells, indicating that SMIE cells are more permeable than MDCK cells. These results suggest that claudin-4 expression contributes to barrier function of intact salivary gland duct cells. [J Physiol Sci. 2006;56 Suppl:S122]

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In order to examine the redox state of cerebrospinal fluid (CSF) albumin together with serum albumin from orthopedic patients, we have analyzed the percentage of reduced and oxidized albumin fractions in CSF and serum by HPLC. Forty-two patients had no detectable evidences of systemic diseases such as hepatic and renal dysfunctions, and they were divided into two groups by their age (young group, average age = 31.3 years (n = 10); elder group, 64.8 years (n = 32)). Informed consent was obtained in all cases. Albumin is the mixture of reduced and oxidized albumins. Oxidized albumin is composed of two type of albumin, i.e., mixed disulfide with cysteine (tentatively called HNA-1) and oxidation product higher than mixed disulfide with reactive oxygen species (called HNA-2). In the elder patients, mean values for the fraction of HMA, HNA-1 and HNA-2 were 93.1, 5.8 and 1.1% for CSF, and 69.5, 28.6 and 1.9% for serum, respectively. In the young patients, those values were 93.0, 6.7 and 0.3% for CSF, and 76.4, 22.0 and 1.6% for serum, respectively. CSF is believed to maintain the homeostasis of brain functions, especially defense against oxidative stress. From our HPLC results, in both groups, significant difference between the values for CSF and serum indicates the function of blood-brain barrier is maintained. Significant high value for reduced albumin level in CSF in both groups indicates the redox state of CSF is kept highly reduced status. [J Physiol Sci. 2006;56 Suppl:S122]

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Long-Evans Cinnamon (LEC) rats have been used as an animal model for human Wilson's disease. LEC rats developed hepatic abnormalities due to accumulation of copper in the liver. The aim of this study was to search for liver proteins related to Wilson's disease. The livers from LEC and WKAH/Hkm rats (12 and 16 weeks of age) were homogenized and performed using two-dimensional electrophoresis (2-DE). Their 2-DE spots were analyzed by an image analysis software for comparison of protein quantity. Although no significant difference was observed between the two strain rats of 12 weeks, 17 spots were different between the two of 16 weeks. One spot of 36.5 kDa was positive in the WKAH rat, but was negative in the LEC rat. Four spots were denser by 1.5 times in the LEC than in the WKAH, Two of them were denser by around 7 times. On the other hand, 8 of 12 spots were much thinner by 2 times in the LEC than in WKAH. These results suggest that two-dimensional electrophoresis may be useful for the study of Wilson's disease. [J Physiol Sci. 2006;56 Suppl:S122]

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We have demonstrated for the first time that the Hemagglutinating virus of Japan-envelope (HVJ-E) vector allows the efficient transduction of protein into the rat brainstem using the technique of microinjection in vivo. Successful transduction of an exogenous protein, β-galactosidase (β-gal), was performed via the direct injection of only 100 nl HVJ-E vector solution into the specific restricted brain area (nucleus tractus solitarius, NTS). To examine whether the β-gal activity was maintained in the rat brainstem, samples were collected 3, 6 and 24 hours after transduction and a colorimetric assay was utilized to detect and quantify β-gal activity. A constant and high transduction level of β-gal activity was maintained in the rat brainstem, that was not significantly reduced within 24 hours following transduction compaired with β-gal without HVJ-E vector. This kind of targeted delivery system using the HVJ-E vector should have wide applications of various therapeutic proteins to the central nervous system in vivo. [J Physiol Sci. 2006;56 Suppl:S122]

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There are three isoforms in Na/K-ATPase α subunit, α1, α2 and α3. It is known that α1 is expressed in wide range of cells/tissues and that α2 and α3 localize abundantly in neuronal tissues. We examined the expression of Na/K-ATPase α isoforms in salivary glands by RT-PCR. Alpha1 was expressed in three major salivary glands. Alpha2 was expressed in submandibular gland (SMG) and sublingual gland (SLG) and faintly parotid gland (PG). Alpha3 was expressed in SLG alone. The DNA sequence of α2 and α3 PCR products from salivary glands were identical with the corresponding portion of brain. We examined mRNA levels of these α isoforms by comparing the quantity of the PCR products. Alpha1 levels in 3 salivary glands were same as that in brain. Alpha2 levels in SMG, SLG and PG were determined to be 1/32, 1/16 and 1/256 of that in brain, respectively. Alpha3 level in SLG was at 1/8. Since α3 is abundant in nerve tissues, we examined whether α3 expression is induced when pheochromocytoma PC12 cells are differentiated into neuron-like cells by nerve growth factor (NGF). PC12 cells expressed α1 and α3, however α3 was not changed by the NGF-treatment of the cells. The α isoforms of HSY cells, a cell line of human parotid gland adenocarcinoma, were also examined. HSY cells expressed α1 alone. The expression pattern of Na/K-ATPase α isoforms in cells/tissues seemed to be rather stable and it was not easily altered with differentiation factors or carcinogenesis. [J Physiol Sci. 2006;56 Suppl:S123]

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In the present study,to investigate the role of apical Na⁺-H⁺ exchangers (NHE) in HCO₃⁻ secretion from pancreatic duct cells, we examined the activity of apical Na⁺-H⁺ exchange in interlobular pancreatic duct segments isolated from normal mice and δF508 mice where function of CFTR (cystic fibrosis transmembrane conductance regulator) is disrupted.Interlobular duct segments were isolated by microdissection.The ducts were superfused with HCO₃⁻-free Hepes-buffered solutions and the lumen was microperfused separately.Intracellular pH (pH_i) was measured by microfluorometry in ducts loaded with pH-sensitive fluoroprobe BCECF.The duct cells were acid-loaded with 20 mM NH₄⁺,which was followed by a Na⁺-free solution in both the bath and lumen.The rate of pH_i recovery after re-addition of Na⁺ to the luminal solution was calculated as a measure of the activity of apical Na⁺-H⁺ exchange.The rate of pHi recovery (dpH/dt) was 0.12 ± 0.01 pH unit/min (mean ± SD, n = 8) in wild type ducts, which was completely inhibited by 100 μM HOE642,an inhibitor of NHE. Forskolin reduced the apical NHE activity to 0.05 ± 0.01 pH unit/min (n = 9, p < 0.01).The apical NHE activity in cystic fibrosis ducts was 0.20 ± 0.01 pH unit/min (n = 6),which was significantly (p < 0.01) higher than that in wild type ducts and was accelerated to 0.66 ± 0.11 pH unit/min (n = 6, p < 0.01) by application of forskolin. In mice pancreatic duct cells,the activity of apical NHE was suppressed by functional CFTR and it was stimulated by cAMP in the absence of functional CFTR. [J Physiol Sci. 2006;56 Suppl:S123]

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Bicarbonate secretion in mouse ileum has at least two components, one being Cl⁻-dependent, and the other being Cl⁻-independent and activated by cAMP. We have previously demonstrated and characterized Cl⁻/HCO₃⁻ exchange activity in the mouse ileal villous cell by microfluorometric intracellular pH (pH_i) measurements. The purpose of this study is to determine if the Cl⁻/HCO₃⁻ exchanger that we characterized is responsible for the ileal Cl⁻-dependent HCO₃⁻ secretion. To this end isolated mouse ileum was mounted in Ussing chamber and the alkaline secretion rates (J_OH(sm)) were determined by continuously titrating the luminal buffer-free solution to pH 7.2 with 1mM H₂SO₄ using a pH stat device. The serosal side was always bathed with HCO₃⁻/CO₂-buffered solution. The Cl⁻-dependent J_OH(sm) was inhibited by 30μM niflumic acid (by 25%) and by 100μM acetazolamide by (40%) both being added to the mucosal side, whereas cAMP-induced J_OH(sm) was not inhibited by these compounds. Glibenclamide (100μM) added to the mucosal side had no effect on either component. Br⁻, I⁻, NO₃⁻, and SO₄²⁻ added to the mucosal side induced J_OH(sm) of 80%, 50%, 50%, and 65%, respectively, of the J_OH(sm) induced by Cl⁻. These inhibitor sensitivity and ion selectivity profiles of Cl⁻-dependent bicarbonate secretion generally agreed with those of the Cl⁻/HCO₃⁻ exchanger we have previously shown in the enterocytes by pH_i measurement, suggesting that the Cl⁻/HCO₃⁻ exchanger is responsible for Cl⁻-dependent bicarbonate secretion. The molecular identity of the exchanger remains to be determined. [J Physiol Sci. 2006;56 Suppl:S123]

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GABA, a major inhibitory neurotransmitter in the adult CNS, is excitatory at early developmental stages due to the elevated intracellular Cl⁻ concentration ([Cl⁻]_i). This functional switch is primarily attributable to the K⁺-Cl⁻ cotransporter KCC2, the expression of which is developmentally regulated in neurons. Previously we reported that KCC2 interacts with brain-type creatine kinase (CKB). To elucidate the functional significance of this interaction, HEK293 cells were transfected with KCC2 and glycine receptor α2 subunit, and gramicidin-perforated patch-clamp recordings were performed to measure the glycine reversal potential (E_gly), giving an estimate of [Cl⁻]_i. KCC2-expressing cells displayed the expected changes in E_gly following alterations in the extracellular K⁺ concentration ([K⁺]_o) or administration of an inhibitor of KCCs, suggesting that the KCC2 function was being properly assessed. When added into KCC2-expressing cells, dominant-negative CKB induced a depolarizing shift in E_gly and reduced the hyperpolarizing shift in E_gly seen in response to a lowering of [K⁺]_o) compared to wild-type CKB. Moreover, 2,4-dinitrofluorobenzene (DNFB), an inhibitor of CKs, shifted E_gly in the depolarizing direction. In primary cortical neurons expressing CKB, the GABA reversal potential was also shifted in the depolarizing direction by DNFB. Our findings suggest that in the cellular microenvironment, CKB activates the KCC2 function. [J Physiol Sci. 2006;56 Suppl:S124]

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Background: Recent studies have revealed that tachyarrhythmias cause electrical and structural remodeling of cardiomyocytes, and that apoptosis contributes to myocardial remodeling in certain arrhythmias. We suspected that tachyarrhythmias lead to arrhythmogenic substrates through the induction of apoptosis, and then may tend to initiate and perpetuate themselves. In this study, we tested the hypothesis that rapid electrical stimulation of cardiomyocytes in culture causes apoptosis. Methods and Results: The cultured H9c2 cardiac cells were subjected to rapid electrical stimulation for 72 hours at a pacing frequency of 5 Hz. To determine the degree of apoptosis, the percentage of hypodiploid cells, mitochondrial transmembrane potential (ΔΨ_m), and activities of caspases-3, -8, and -9 were measured quantitatively. Compared with the cells in the absence of stimulation, the cells subjected to rapid stimulation showed apoptosis in terms of the appearance of hypodiploid cells (4.7±0.5% vs 31.0±8.5%), loss of ΔΨ_m (5.3±0.5% vs 19.5±2.7%), and activation of all caspases tested (caspase-3, 0.050±0.015 vs 0.117±0.030; caspase-8, 0.038±0.004 vs 0.056±0.002; caspase-9, 0.029±0.003 vs 0.046±0.010) with statistically significant differences. Conclusions: Rapid electrical stimulation of H9c2 cells induced apoptosis via both the death receptor and mitochondrial pathways. The present in vitro model may be useful to elucidate mechanisms of tachycardia-induced myocardial remodeling via apoptosis, and to lead to therapies for preventing the arrhythmogenic substrate development. [J Physiol Sci. 2006;56 Suppl:S124]

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The ionic mechanisms underlying pacemaker activity have been extensively studied in the sinoatrial (SA) node, but little is known about the molecular basis of ionic currents. The aim of this study was to evaluate mRNA expression for ion channels, connexins and Ca²⁺-handling proteins using quantitative PCR and to visualize the distribution of these transcripts using in situ hybridization in and around the rabbit SA node. Quantitative PCR showed that there were significant differences in the abundance of 60% of the 30 mRNAs studied in different tissues (SA node center, SA node periphery and atrial muscle). Grouping analysis of the PCR data identified two significantly different clusters: mRNAs tended to increase (Cx45, Nav1.1, Cav1.3, HCN1, HCN4, Kv4.2, ERG, KvLQT1, Kir2.2 and Kir3.1) and those tended to decrease (Cx43, Cx40, RYR2, SERCA2a, Nav1.5, Cav1.2, Kv1.4, Kv4.3, KChIP2, Kv1.5, minK, Kir2.1 and Kir6.2) from the atrial muscle to the center of the SA node. The mRNA expression profiles of the center and the periphery of the SA node were similar but there were significant differences for some transcripts (e.g. Cav1.3, HCN1 and HCN4). In situ hybridization confirmed these regional differences in the mRNA expression pattern. This study shows a complex variation in the expression of ion channel mRNAs from the atrial muscle to the SA node, which may be important in the functional organization of the SA node as a physiological and dependable pacemaker of the heart. [J Physiol Sci. 2006;56 Suppl:S124]

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We investigated whether the alteration of Ca²⁺ uptake rate in sarcoplasmic reticulum (SR) would influence the Ca²⁺ content and the leakage of Ca²⁺ in SR. In this study, we used saponin-treated thin trabeculae obtained from the hearts which overexpresses sarcolipin (SLN). We reported that SLN reduced the activity of SR Ca²⁺-ATPase. The advantage of saponin-treated preparation is that integrated structure around SR and Ca²⁺ handling functions are well preserved. Thus, the functions of SR to release and uptake Ca²⁺ can be measured under more physiological conditions compared to isolated vesicular SR. Ca²⁺ content was measured by releasing all Ca²⁺ from SR by caffeine (50 mM) after loading Ca²⁺ (pCa 6.2) into SR in the presence of ATP (4 mM) for different periods. Leaked Ca²⁺ was estimated by measuring the remaining Ca²⁺ in SR after washing for various durations, following Ca²⁺ loading. Ca²⁺ uptake rate was slower in SLN transgenic (SLN-TG) than that in non-transgenic (SLN-NTG) myocardium. The maximal Ca²⁺ content and leakage of Ca²⁺ in SLN-TG and SLN-NTG were almost identical. The results suggest that the modulation of Ca²⁺ uptake rate could not affect maximal Ca²⁺ content and Ca²⁺ leakage measured at steady state. The modulation of Ca²⁺ uptake rate influences Ca²⁺ handling in each heartbeat but does not alter the maximal Ca²⁺ content and leakage at steady state. [J Physiol Sci. 2006;56 Suppl:S125]

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To understand the pathophysiology of hereditary cardiomyopathy, we measured the phosphorylation status of contractile proteins, ventricular light chain 2 (VLC2), troponin I (TnI), troponin T (TnT) and myosin-binding protein C (MyBP-C), and the Ca²⁺-dependence of tension and ATPase activity in skinned trabeculae obtained from normal control (F1B) and cardiomyopathic hamsters (TO-2). To change the phosphorylation status of contractile proteins, isolated trabeculae were exposed to Tyrode's solution containing 30 mM BDM for 30 minutes before skinning. In the BDM-untreated preparations, all the contractile protein phosphorylation levels were higher in F1B than in TO-2, while the Ca²⁺ sensitivities of tension and ATPase activity were substantially lower in F1B than in TO-2. The BDM treatment did not alter the contractile protein phosphorylation levels as well as the Ca²⁺ sensitivities of tension and ATPase activity in TO-2 preparations. However, the BDM treatment decreased the contractile protein phosphorylation levels as well as increased the Ca²⁺ sensitivities of tension and ATPase activity in F1B preparations to the levels similar to those in TO-2 preparations. These results suggest that the increase in Ca²⁺ sensitivities of tension and ATPase activity in TO-2 hamster hearts results from the decreased basal level of TnI and TnT phosphorylation, since the dephosphorylation of VLC2 and MyBP-C has been reported to decrease Ca²⁺ sensitivities. [J Physiol Sci. 2006;56 Suppl:S125]

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Background: Recently, mutations of the phospholamban gene, a peculiar inhibitor of sarcoplasmic reticulum calcium ATPase (SERCA2a), have been found in patients with familial cardiomyopathy. This result suggests that mutations of SERCA2a gene also cause cardiomyopathy. In our previous study, we found one patient with hypertrophic cardiomyopathy who harbored a missense mutation in the SERCA2a gene. The mutation changed the amino acid sequence from valine (540) to alanine in the cytoplasmic region close to the ATP binding site. Objective: We examined whether the V540A mutation of SERCA2a could cause the abnormal cardiac function or not. Methods: Mouse SERCA2a cDNA was cloned and the V540A mutation was inserted into it. Transgenic mice overexpressing the V540A mutation of SERCA2a in the heart was generated. Cardiac phenotypes were observed in SERCA2a transgenic mice (TG) using in vivo echocardiography and hemodynamic analyses. Results: By Western blot analysis, we found that the expression of SERCA2a protein was increased by 1.7-fold in the ventricles of TG when compared with those of non-transgenic mice. The weight of the left ventricle was slightly, but significantly increased in TG. The maximal first derivate of left ventricular pressure in TG was significantly lower by 16%, when compared with non-transgenic mice. Conclusion: We concluded that the mutation of the SERCA2a gene resulted in the abnormal cardiac function in mice. Our results imply that the V540A mutation of the SERCA2a gene is a causal mutation in human cardiomyopathy. [J Physiol Sci. 2006;56 Suppl:S125]

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In the brain nitric oxide (NO) is implicated in blood flow regulation, neurotransmitter release, learning and memory, neuronal plasticity, and neuronal degeneration and survival. To understand the role of NO in physiological as well as pathological conditions, it is essential to observe the dynamics of NO production itself. Using an NO-selective electrode system that allows the real-time in vivo measurement of NO, we examined changes in NO production during hypoxia and reoxygenation in the striatum of rats from late prenatal to adult stages. Under urethane anesthesia, an NO-selective electrode and laser Doppler flow probe was inserted into the striatum for measuring changes in NO concentration and tissue blood flow, respectively. In adult rats, spontaneous rhythmic production of NO ranging from about 0.2–1.5 Hz was observed, which was not related directly to cardiac or respiratory cycles. The rhythmicity in NO production was reduced after systemic administration of the NMDA channel blocker ketamine. In adult rats, brain hypoxia abolished the rhythmicity, but caused a large amount of NO production in the striatum. This overproduction of NO during hypoxia was also observed in the fetal and neonatal rat brain, although the basal rhythmic NO production was not detected. The immature rat brain may have less capacity to produce NO than the matured rat brain. The appearance of the rhythmic NO production in the brain may indicate the maturity of the brain NO-producing system. Further study is needed to clarify the origin of this rhythmic NO production in the rat brain. [J Physiol Sci. 2006;56 Suppl:S125]

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Multiple-site optical recording methods, using a fast voltage-sensitive merocyanine-rhodanine dye (NK2761) and a multi-element (16 X 16) photodiode array, were employed to simultaneously monitor action potentials from many sites in the isolated rat atrium preparation. Cytochalasin D was used to reduce contraction-related optical artefacts by suppressing muscle contraction without affecting electrical action potential. The suppression effect of this chemical (40μM) on the contraction-related optical artefacts is time-dependent for the first 30 min. and become steady state within 40–60 min. after the application to the bath. The suppression of the artefact of this chemical is generally stronger than that of 2,3-butanedione monoxime (BDM: 20 mM). Using this chemical, we successively evoked tachycardia-like excitation (TE) in the isolated rat right atrium preparation and mapped optically the excitation spread pattern during TE. [J Physiol Sci. 2006;56 Suppl:S126]

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Cardiac Na⁺/Ca²⁺ exchanger1 (NCX1) expression levels change under various pathophysiological conditions, e.g. heart failure. However, its mechanism is unknown. We previously showed that fluvastatin (Flv), an HMG-CoA reductase (HMGR) inhibitor, decreased NCX1 mRNA and protein expression via inhibiting a small G-protein, RhoB in H9c2 cardiomyoblasts. Flv-induced down-regulation of NCX1 mRNA was reversed by mevalonate, farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP), suggesting an involvement of isoprenylation of RhoB. Conversely, we also found that lisophosphatidylcholine (LPC) increased NCX1 mRNA and protein by activating RhoB. RhoB requires isoprenylation for its activation by either GGPP or FPP. Here, we investigated which isoprenoid is involved in NCX1 increasing effect of LPC. Treatment of H9c2 cells with Flv for 24 hours decreased NCX1 mRNA to about 60% of control. Addition of GGPP or FPP restored NCX1 mRNA, which had been decreased by Flv, to a control level within 24 hours. No significant difference was observed between GGPP and FPP. When LPC was applied with Flv, NCX1 mRNA remained decreased. However, when LPC and GGPP were applied simultaneously, NCX1 mRNA was increased to a level significantly higher than the control. Unlike GGPP, FPP did not induce this increase. These results suggest that an isoprenoid involved in the effect of LPC increasing NCX1 mRNA is GGPP but not FPP. [J Physiol Sci. 2006;56 Suppl:S126]

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Background: It is postulated that T-type Ca²⁺ channels play important roles not only in physiological condition of various organs and tissues, but also during the progression of various diseases. In this study, we tested the hypothesis that Ca²⁺ entry through the T-type Ca²⁺ channels causes apoptosis using recombinant Ca_V3.2 (α1H) T-type Ca²⁺ channels expressed in HEK293 (HEK-α1H) cells. Methods and Results: Cultured HEK293 cells and HEK-α1H cells were incubated for 12 hours in serum-free medium containing different concentrations of Ca²⁺ (1.8-7.2 mM). To determine the degree of apoptosis, mitochondrial transmembrane potential (ΔΨ_m) and activities of caspase-3, -8, -9 were measured quantitatively. Intracellular free Ca²⁺ measured by flow cytometry using Fluo-3 was elevated depending on Ca²⁺ concentration in the medium ([Ca²⁺]o) in HEK-α1H cells but not in HEK293 cells. In HEK-α1H cells, apoptosis in terms of loss of ΔΨ_m and activation of caspase-3 and -9 was observed at [Ca²⁺]o of 5.4 mM or more, while caspase-8 was not activated. In contrast, apoptosis was not induced at any [Ca²⁺]o in HEK293 cells. Conclusion: We demonstrated that Ca²⁺ entry through the T-type Ca²⁺ channels causes apoptosis via the mitochondrial pathway. Thus, T-type Ca²⁺ channels may be therapeutic targets for certain pathophysiological conditions related to apoptosis. [J Physiol Sci. 2006;56 Suppl:S126]

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Dysfunctions in the gating of ryanodine-receptors (RyR) and intracellular Ca²⁺ handling play critical roles to evoke lethal arrhythmias not only in inherited arrhythmgenic syndromes such as CPVT, but also in more common cases including heart failure. To develop a human ventricular action model which allows analyses of the effects of altered RyR properties on arrhythmogenesis, we incorporated a calcium release system with 4-state Markovian RyR gating (Stern, J.Gen.Physiol.1999) into a simplified human action potential model (TenTusscher, Am.J.Physiol.2004). Introduction of Ca concentration-dependence on RyR opening rate was a first step to model Ca-induced Ca-release. To obtain reasonable RyR gating in the control, it was necessary to add a new Ca compartment (cleft), where Ca concentration increased and decayed much faster than cytosolic Ca transient. Under these settings, augmented sensitivity of RyR on cleft Ca easily evoked spontaneous oscillatory Ca releases during the decay phase of Ca transient. Induction of DADs leading to triggered activity, however, could not be reproduced straightforwardly in spite of further inclusion of SR Ca-load dependence of RyR gating. Generation of triggered action potential after repolarization required additional manipulation of sarcolemmal current systems including a reduction of I_K1, a situation found in patients with terminal heart failure. Our model provides basis to elucidate mechanisms of Ca-mediated arrhythmias, and further to find out RyR-targeted anti-arrhythmic therapy. [J Physiol Sci. 2006;56 Suppl:S126]

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To characterize Ca²⁺handling by the junctional sarcoplasmic reticulum (JSR) in heart muscle of hypertensive rat(SHR), we examined the time course of short-term mechanical restitution(STMR) after varying magnitude of twitch contraction with the papillary muscle of adult(20 weeks-old)SHR and age-matched control(WKY) and action potential duration at various stimulus frequencies. The slope of the STMR in WKY was independent of the magnitude of the preceding twitch and similar to that of SHR unless the preceding twitch was not so large as the rested state twitch. The slope decreased after the rested state twitch in SHR. The functions G(t) and H(t), representing the time courses of the JSR Ca²⁺ replenishment and release, respectively, were derived graphically from a family of the mechanical restitution curves for the both strains. The action potential duration as measured at its half width was significantly greater in SHR than WKY(p <0.005). Both a time to peak tension and a half relaxation time were significantly longer in SHR than WKY(p <0.0001). The result suggests slower Ca²⁺ replenishment to the JSR in SHR than WKY irrespective of the longer action potential in the former than the latter. [J Physiol Sci. 2006;56 Suppl:S127]

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To clarify complex interactions between cardiac excitation-contraction (E-C) coupling and energy metabolism, a cardiac mitochondria model is developed, which consists of oxidative phosphorylation, TCA cycle, pyruvate metabolism, fatty acid β oxidation, malate shuttle and Ca²⁺ dynamics. Using this model, we studied regulation of mitochondrial function by Ca²⁺, and investigated contribution of each functional element of oxidative phosphorylation to mitochondrial oxygen consumption (mVO2), mitochondrial membrane potential and NADH. Calculation of the membrane potential was improved by integrating charge flux across the membrane via proton pumps and transporters. The model simulation revealed that the activation of F1F0-ATPase, adenine nucleotide translocator enhanced mVO2 by about 60%, This result is supported by experimental data demonstrating a large increase in mVO2 of state 3 mitochondria by Ca²⁺ (Territo et al. Am J Physiol. 278:C423-35, 2000). The expanded computer model, which combines the mitochondria model with an E-C coupling model of cardiomyocyte (Kyoto model), suggested that Ca²⁺-dependent activation of both oxidative phosphorylation and Ca²⁺-dependent dehydrogenases plays pivotal roles in regulating cardiac mitochondrial function by stabilizing metabolite concentrations during an increase in workload induced by changing beating frequency. [J Physiol Sci. 2006;56 Suppl:S127]

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Acetaldehyde (AcA), an oxidized product of alcohol, may contribute to alcohol-induced cardiac muscle dysfunction. Recently, we have demonstrated that even at 1-3 μM, AcA activates the ryanodine receptor type 2 (RyR2) channel and promotes Ca²⁺ release from the cardiac muscle SR (Oba et al., JJP 55:S90, 2005). AcA produces ROS and oxidizes GSH to GSSG to lead to oxidative stress in tissues. We studied whether cytoplasmic redox potential (RP) affects AcA-induced activation of the RyR2 channel and mediates cardiac dysfunction. In experiments under RP control using glutathione buffers, fixation of cytoplasmic (cis) RP at the oxidative state activated the channel incorporated into bilayers, whereas definition of luminal (trans) potential did not. Exposure of the RyR2 to AcA without defining RP stimulated markedly the channel in a dose-dependent manner (1-100 μM). When RP in cis/trans sides of the channel was fixed at -220/-180 mV, AcA at concentrations less than 3 μM caused no longer the channel activation, but higher concentrations increased 2 fold the open probability. In condition under cis/trans potential at -250/-180mV, AcA failed to activate the RyR2 channel. These results indicate that the activation of RyR2 channels by AcA exposure is elicited even at reductive states and suggest that cytoplasmic RP may have a protective effect against pathophysiological changes by AcA in cardiac myocytes. [J Physiol Sci. 2006;56 Suppl:S127]

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Purpose: We have recently reported that the effects of α₁-adrenoceptor stimulation (α₁ARS) on L-type Ca²⁺ current (I_Ca) can be classified in two opposite effects (negative and positive effects) and the positive effect is protein kinase C (PKC)-dependent. We postulate that these two effects simultaneously occur through different subtypes of α₁-adrenoceptor and different intracellular signal transduction pathways. In this study, we investigated the effects of α_1AARS and α_1BARS on I_Ca. Methods: Perforated patch-clamp was used for recording of I_Ca from isolated adult rat ventricular myocytes. Holding potential was set at -40 mV and depolarization pulse to 0 mV was applied every 10 sec. Results: Biphasic change in I_Ca (a transient decrease followed by a sustained increase) was induced by a non-selective α₁-adrenoceptor agonist, phenylephrine (Phe). However, a selective α_1A agonist, A61603 showed only positive effect on I_Ca, and the application of Phe with a selective α_1A antagonist, WB4101, caused only sustained negative effect. After pertussis toxin treatment, a transient decrease in I_Ca induced by Phe disappeared and only sustained increase was observed. Conclusion α_1AARS showed positive effect on I_Ca, which was PKC-dependent. On the other hand, α_1BARS showed negative effect mediated through G_i/o protein. Thus, α_1AARS and α_1BARS produce opposite effects on I_Ca via different intracellular signal transduction pathways. [J Physiol Sci. 2006;56 Suppl:S127]

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