ATP1AL1 is a human nongastric H⁺,K⁺-ATPase. The physiological function of ATP1AL1 has not been clarified yet. Herein we have cloned a wild type and a novel splicing valiant deleting exon 4 of ATP1AL1 from human colorectum. Then, the stable cell lines expressing gastric H⁺,K⁺-ATPase β-subunit were transfected with the pcDNA4/His-ATP1AL1 cDNA (wild type or the variant) construct. Exon4 encodes amino acids 77-144 of ATP1AL1, and this region includes the M1 transmembrane domain and the extracellular loop between M1 and M2. Messenger RNA of the splicing variant deleting exon 4 of ATP1AL1 was expressed in human colorectum, kidney and brain. Protein expression of ATP1AL1 in the cell lines was monitored by using anti-Xpress antibody which reacts with the epitope at the N-terminal of the construct. Interestingly, the variant protein could be expressed in the membrane fraction of the cells. The enzyme activity of ATP1AL1 in the transfecting cells was estimated by subtracting 5 μM ouabain-sensitive activity from 1 mM ouabain-sensitive activity. The variant seems to be ouabain-insensitive, because exon 4 contains the region which is one of the important ouabain-binding sites of Na⁺,K⁺-ATPase. The enzyme activity of both wild type and the variant were sensitive to 100 μM SCH28080, an inhibitor of gastric H⁺,K⁺-ATPase. These results suggest that the splicing variant of ATP1AL1 may be functional in the human colorectum. [Jpn J Physiol 54 Suppl:S80 (2004)]

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Fibroblast is the mesenchymal cell that develops connective tissues of many organs. It can be cultured easily, but it scarcely redifferentiate. As it is known that fibroblast C3H/10T1/2 redifferentiates into muscle cell though in low efficiency, we studied the redifferentiation of various fibroblasts Balb/3T3, C3H/10T1/2, Balb/3T12-3, or C3H/MCA cell line by using MyoD-EGFP chimeric protein expression with transfection of the cDNA. And we also analyzed the qualitative characters of redifferentiation by the expressed MyoD-EGFP chimeric protein with that by MyoD protein derived from a transfected 2-cystronic vector that expressed both MyoD and EGFP respectively. Myogenesis of these mouse embryonic fibroblasts was analyzed by myosine immunochemical staining or L-type Ca2+ channel recording. And we further studied whether 5-azacytidine had some effect on the redifferentiation into myogenic cells. These transfected fibroblasts first fused together and then differentiated into myoblasts. The cell line, that is known to be more tumorigenic such as Balb/3T12-3 or C3H/MCA, fused more effectively than that of Balb/3T3 or C3H/10T1/2. The fused cell, whose original cell line was more tumorigenic, was also differentiated more effectively. It was also observed that DNA demethylation in fibroblast Balb/3T12-3 by 5-azacytidine effectively facilitated the redifferentiation into myotube. The molecular mechanism, that explains the facts that the effective redifferentiation relies on high tumorigenicity and gene demethylation of the fibroblast, must be studied further. [Jpn J Physiol 54 Suppl:S80 (2004)]

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Mitochondrial dysfunction is associated with several diseases such as neuronal degeneration and diabetes, and has also been linked to ageing. Mitochondrial DNA (mtDNA) damages by reactive oxygen species appear to be a major contributor to the dysfunction. To determine mtDNA damages, this study used the combination of long polymerase chain reaction (PCR) and fluorescence correlation spectroscopy (FCS) which is suitable for mass examination. FCS permits the sensitive estimation of the number and the length of PCR products without the electrophoresis. The long PCR assay is based on the assumption that DNA lesions such as strand breaks and base modifications will block the progression of the polymerase. The band of 16 kbp-PCR products on the gel was detected with total DNA isolated from 293 cells. The fragment lengths of the product digested with Eco 47I were same as those predicted from human mitochondrial genome on the database. When the product was stained with YOYO-1, a large fluorescence enhancement was observed due to the intercalation. To monitor the degradation of the labeled DNA with Hae III, FCS measurements were carried out. The number of DNA molecules in volume element increased from 5 to 66, corresponding to fragmentation of the PCR product. The diffusion time derived from Brownian motion of the DNA molecules was also shortened from 19.5 ms to 10.1 ms. These data demonstrate that the mtDNA PCR product is detected by FCS and suggest the feasibility to measure mtDNA damage by oxidative stress. [Jpn J Physiol 54 Suppl:S80 (2004)]

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Effect of extracellular ATP on adipocyte differentiationMariko Omatsu-Kanbe¹, Kazuko Inoue², Norihisa Fujita² and Hiroshi Matsuura¹Dept. Physiol. Shiga Univ. Med. Sci.¹, Otsu and Lab. Bioinformatic Chem., Ritsumeikan Univ.², Kusatsu, Japan.3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Cells of this substrain undergo a preadipose to adipose-like conversion as they progress from rapidly dividing to a confluent and contact inhibited state to stop proliferation. Over the course of several days confluent 3T3-L1 preadipocytes can be converted to adipocytes in the presence of an adipogenic cocktail. It has been recognized that master regulators of differentiation, such as C/EBP and PPARγ, regulate the transcription of adipose-related genes under the cocktail. We investigated whether P2 receptor play a role in 3T3-L1 adipocyte differentiation. In cells of ~30% density, an application of extracellular ATP for 4 min prior to the addition of the cocktail induced adipocyte differentiation, while solely added cocktail did not cause differentiation. In confluent cells, extracelluar ATP did not induce differentiation or did not affect the cocktail-induced differentiation. The effect of ATP was blocked by P2 receptor antagonists suramin and PPADS. ATP-induced differentiated adipocytes contained oil Red O-stained lipid and expressed marker genes of fat cells. The results suggest that P2 receptor play an important role in the adipocyte differentiation without stopping cell proliferation. [Jpn J Physiol 54 Suppl:S81 (2004)]

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AMPA-subtype glutamate receptors (AMPARs), which mediate fast excitatory neurotransmission in most of CNS neurons, are also expressed in glioma cells. To clarify their pathophysiological significance, we used a human glioma cell line U-87MG as a model system. We first examined the expression of AMPAR subunits, GluR1-GluR4, by RT-PCR and immunoblot analyses. This cell line endogenously expressed GluR1, GluR2 and GluR3. Regarding the GluR2 subunit, both the edited form (GluR2R) and unedited form (GluR2Q) were co-expressed in U-87MG cells. Next, we measured AMPA-induced changes in the intracellular Ca²⁺ concentration using fura-2AM. The application of 100 μM AMPA together with 100 μM cyclothiazide significantly increased the Ca²⁺ concentration in ~30% of the total cells tested, and this effect was blocked by an AMPAR antagonist NBQX at 40 μM. When GluR2R was overexpressed using adenoviral vectors, no AMPA-induced rise of the intracellular Ca²⁺ concentration was detected. Furthermore, the GluR2R gene transfer caused retraction of cytoplasmic processes within 4 days after viral infection. These morphological changes did not occur in the presence of 40 μM NBQX in the culture medium. Viability rates significantly decreased in GluR2R-transfered cells, and this was also restored by NBQX. These changes were specific in GluR2R-transferred cells and were never seen when GluR1, GluR3, GluR4 and GluR2Q were transferred. These results indicate that GluR2R plays a critical role in viability and proliferation of U87-MG glioma cells. [Jpn J Physiol 54 Suppl:S81 (2004)]

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Recent studies show that transepithelial ion transport plays an important role in fundamental cellular functions such as cell proliferation and cell death. Particularly, chloride ion transport is reported to affect cell proliferation by regulating cell volume. In addition, mitochondria which have an extremely important role as energy-producing organella are known to be influenced by intracellular ion environments. In the present study, we studied whether chloride ion transport regulates cell proliferation and activity of mitochondria in renal epithelial A6 cells by using regulators of chloride ion channel and Na+/K+/2Cl- cotransporter (NKCC). The cell proliferation was examined by MTS assay and direct cell count. The activity of mitochondria was measured by fluorescent coloring with mito-Red. Treatment with NPPB (a chloride ion channel blocker) and quercetin (an NKCC regulator) inhibited cell growth and activity of mitochondria, but bumetanide (an NKCC inhibitor) did not affect them. Based on these results, it is suggested that regulators of chloride ion transport would influence cell proliferation and mitochondrial activity by regulating intracellular ion environments. Supported by JSPS 15659052 (YM), 15590189 (NN), 15790120 (HM), 15591427 (HI). [Jpn J Physiol 54 Suppl:S81 (2004)]

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The objective of this study is to examine the physiological effects of exposure to ELF magnetic fields on regulation of differentiation in cultured osteoblastic cells (MC3T3-E1) at the growing and the differentiated state. Sinusoidal (60Hz) magnetic fields were about 3 mT. Collagen or non collagen protein contents of cultures were measured microscopically by using ImSpector system. In exposure to ELF for 3 days, both collagen and non collagen protein contents increased with exposure time in the peripheral regions of culture dish than that in the central regions. The treatment of IGF increased in collagen contents in the peripheral cells for 17 days culture. But the additive effects of the exposure and IGF treatment were not observed. On 21 days cells, NGF treatments brought to increase of collagen contents. Then ALP activity was significantly increase by the exposure alone or by combined with NGF treatment. These results indicate that the mechanisms of differentiation related to IGF and NGF in the osteoblasts were altered by the magnetic fields of extremely low frequency. [Jpn J Physiol 54 Suppl:S81 (2004)]

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Microglia are thought to play important functional roles in the brain in both physiological and pathological states, and considered the only cell population of mesodermal origin in the brain, although their role is not fully understood. We recently published a novel role for microglia as multipotential stem cells to give rise to neurons, astorocytes or oligodendrocytes in serum-free medium followed by culture in the presence of high concentration serum. However, we could not observe neuroectodermal differentiation from macrophage cultured in the same condition of microglia (unpublished data). These results raise the possibility that the microglia-specific genes are expressed and functioned during differentiation into neuroectodermal cells. Therefore, we considered that molecular cloning of the differentiation-specific factors would provide a clue to understanding the unrevealed molecular mechanisms, which underlie complex signaling pathways of neuroectodermal differentiation from microglia. For this purpus, we performed a subtraction experiment using mRNA isolated from microglia and macrophage, cultured in the differentiated condition as described above. We detected 3 novel genes and some interest genes involved in neural functions. We now characterize their roles in neuroectodermal differentiation of microglia, and continue to screen other genes by this method. [Jpn J Physiol 54 Suppl:S82 (2004)]

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Background. Although inhibition of angiogenesis represents a beneficial approach for the treatment of diseases such as cancer and diabetic retinopathy, knowledge about intrinsic angioinhibitory molecules has been limited. Sphingosine-1-phosphate (S1P) mediates pleiotropic effects in diverse cell types through Edg family G protein-coupled receptors. Interestingly, S1P exerts an inhibitory action on cell migration via Edg-5 receptor subtype. We studied whether S1P/Edg-5 system could inhibit tube formation and migration in vascular endothelial cells. Methods and Results SVEC4-10, a vascular endothelial cell line derived from mouse lymph node, possesses the ability to form tube-like structures on the Matrigel. Northern blot analyses revealed that SVEC4-10 expresses Edg-5 and 3, but not Edg-1. S1P weakly stimulated tube formation and the addition of JTE-013, a specific antagonist against Edg-5, enhanced S1P-induced tube formation. Similarly, JTE-013 potentiated S1P stimulation of cell migration, assessed by a modified Boyden chamber method. In the cells stably overexpressing Edg-5 (SVR-5), S1P markedly inhibited epidermal growth factor (EGF)-induced tube formation and migration in dose-dependent manners and the inhibition was completely reversed by JTE-013. S1P inhibited EGF-induced cellular Rac activation in SVR-5 cells, and JTE-013 cancelled the S1P inhibition. These results suggest that S1P/Edg-5 system plays certain roles in the regulation of tube formation and migration in the cells and might be applicable to angiostatic therapy. [Jpn J Physiol 54 Suppl:S82 (2004)]

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We previously isolated a cDNA encoding a novel large GTP-binding protein (mOPA1) from the mouse brain. In transfected cultured cells, mOPA1 is localized to mitochondria and the overexpression of mOPA1 induced dramatic morphological changes of mitochondria from a tubular to a fragmented shape.
We have identified that the C-terminal region of mOPA1 intensely interacted with each other using a yeast two-hybrid method. In this study, we tried to confirm the intracellular homomeric interaction of mOPA1. FLAG- and Myc-tag sequence were added to the C-terminus of wild type mOPA1 (960 a.a. residues) or C-terminal deletion mutant of mOPA1 (525 a.a. residues). COS-7 cells were cotransfected with both tagged mOPA1, and the immunoprecipitation was carried out using anti-FLAG antibody from cell extracts. We confirmed that Myc-tagged mOPA1 was coprecipitated with FLAG-tagged mOPA1, indicating that wild type mOPA1 was homomultimerized in transfected cells. Unexpectedly, C-terminal deletion mutant of mOPA1 was also coprecipitated although the intense interaction domain was removed.
In transfected COS-7 cells, C-terminal deletion mutant of mOPA1 showed a vesicular distribution in mitochondria, but mitochondrial fragmentation was not observed. These results suggest that homomultimerization of mOPA1 is not sufficient and the C-terminal sequence of mOPA1 is necessary for mitochondrial fragmentation. [Jpn J Physiol 54 Suppl:S82 (2004)]

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Real-time imaging has revealed that mitochondria exhibit dynamic movement and morphological changes in living cells. However, the regulatory mechanisms involved in these processes and their physiological significance are unclear. We showed that in cultured hippocampal neurons distinctive mitochondrial dynamics and morphological changes were regulated by channel specific Ca²⁺ signaling and these changes control neuronal survival and death. VDCC -associated Ca²⁺ elevation induced by high potassium stimulation triggered immediate cessation of movement and late onset of ring-like morphological changes. By contrast, NMDA receptor-associated Ca²⁺ signaling induced by glutamate stimulation produced a relatively slow onset of movement ceasing and the elongated mitochondria become round in morphology. Glutamate-induced mitochondrial change was accompanied by cytochrome c release and led to excitatory neuronal death. These changes were blocked by calcineurin inhibitors FK506 and cyclosporin A. On the other hand, the VDCC-associated changes appeared to be resistant to the cell death. CaMKI-dependent mitochondrial division and increase of mitochondrial volume contributed to the protective effect. These results demonstrated that mitochondrial dynamics and morphological changes are regulated by channel specific calcium signals and such changes exert critical impact on neuronal functions. [Jpn J Physiol 54 Suppl:S82 (2004)]

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Mitochondria play extensive roles in cells by performing dynamic movement and morphological changes. Mitochondria maintain the elongated morphology and appropriate volume by the balance of fusion and division. However, it is unclear how mitochondria perform morphological changes in response to the external stimulation. We have investigated the intracellular signaling pathways that regulate the mitochondrial morphology in cultured hippocampal neurons. Mitochondria were labeled by expressing mitochondria-targeted fluorescent protein and their movement and morphology were monitored with real time imaging. High potassium stimulation induced a division-like changes and cessation of movement. This effect was dependent on the activation of N and P/Q types of voltage-dependent Ca²⁺channel (VDCC). A Ca²⁺/Calmodulin-dependent kinase inhibitor, KN93 blocked this change, but KN92 had no effect. Expression of dominant negative CaMKI blocked the mitochondrial response to potassium stimulation. On the other hand, glutamate stimulation induced the slow onset of movement freezing and morphological contraction. These changes were dependent on the activation of NMDA receptors and calcineurin signaling pathway. These results demonstrated that mitochondrial dynamics and morphological changes are regulated by distinctive intracellular signaling pathways associated with specific calcium channels. [Jpn J Physiol 54 Suppl:S83 (2004)]

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Apoptosis is essential for normal tissue development and homeostasis. The apoptotic volume decrease (AVD), which is induced by KCl efflux due to activation of K⁺ and Cl⁻ channels, is an early-phase, prerequisite component of apoptosis. We previously demonstrated that volume-sensitive Cl⁻ currents are activated after application either of a mitochondrion-mediated inducer, staurosporine (STS), or of a death receptor-mediated inducer, TNF-α or Fas ligand, in HeLa cells without showing cell swelling. Here, we investigated a role of reactive oxygen species (ROS) in STS-induced activation of Cl⁻ channel. STS was found to increase the intracellular ROS level by using 2', 7'-dichlorofluorescein diacetate. The rise in ROS was inhibited by a ROS scavenger, N-acetyl-cysteine (NAC), or an NAD(P)H oxidase inhibitor, diphenylene-iodonium chloride (DPI). In the presence of NAC or DPI, STS failed to activate Cl⁻ currents. In addition, extracellular application of hydrogen peroxide directly increased Cl⁻ currents, which exhibited properties identical to those of volume-sensitive Cl⁻ currents. NAC and DPI could abolish the AVD induced by STS. STS-induced caspase-3 activation and reduction of cell viability were also suppressed by NAC and DPI. These results suggest that ROS is a key mediator for activation of volume-sensitive Cl⁻ channel during a mitochondrion-mediated apoptosis. [Jpn J Physiol 54 Suppl:S83 (2004)]

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Jasplakinolide, a cell-permeable monocyclic peptide, binds to the F-actin and induces actin stabilization in vitro. In epithelial cells, actin filament network is important for the functions, because disruption of actin filament network causes morphological alteration, dissociation of mitosis and cell death. In salivary glands, actin filaments have been suggested to contribute to cell-cell adhesion, polarization and scaffold of plasma membrane. Apoptosis is a regulated process by which a cell undergoes a form of cell death characterized by DNA cleavage, nuclear condensation, cell shrinkage and membrane blebbing. We here demonstrate that jasplakinolide induces apoptosis in rat parotid acinar cells. Parotid acinar cells of male Sprague-Dawley rats were dispersed using trypsin and collagenase digestion. For morphological study, confocal and conventional transmission electron microscopies were used. DNA ladder was electrophoretically separated. When parotid acinar cells were treated with jasplakinolide, decrease of fluorescence intensity of submembrous F-actin was observed in fluorescence microscopy. In the cells, actin filament aggregation was elicited. In conventional transmission electron microscopy, chromatin aggregation, membrane blebbing and apoptotic bodies were observed exclusively in the jasplakinolide-treated cells. Jasplakinolide-induced apoptosis was confirmed by detection of DNA ladder and DNA fragmentation. Taken together, jasplakinolide induces apoptosis in rat parotid acinar cells. [Jpn J Physiol 54 Suppl:S83 (2004)]

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Purpose: Rare sugars are the class of monosaccharides and their derivatives are rare in nature. There are about 50 kinds of rare sugars and one of them is D-psicose. Recent characterization of physiological function of these sugars revealed a novel inhibitory effect on production of reactive oxygen species (ROS). The ROS leads to oxidative stress causing a cell injury including neuronal cells, inducing cell death via apoptosis. We examined the neuroprotective effects of D-psicose on 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells, the in vitro model of Parkinson's disease. Material and Methods: Apoptotic characteristics of PC12 cells were assessed by MTT, TUNEL and DNA fragmentation assay. Results: The 50 mM D-psicose exerted significant protective effects against 6-OHDA(200μM)-induced apoptosis, while other sugars such as D-fructose and D-glucose had insignificant or no protective effects. We noticed a significant elevation in intracellular glutathione level at 24 hours after 6-OHDA treatment, while a decrease was noticed at 3 and 6 hours. Especially, exposure with D-psicose and 6-OHDA for 24 hours showed higher intracellular glutathione levels. Conclusions: The increased intracellular glutathione level may well protect the cell from apoptosis by decreasing the oxidative stress and increase cell viability. Our results show that D-psicose may play an important role as a neuroprotective agent for the neurodegenerative diseases. [Jpn J Physiol 54 Suppl:S83 (2004)]

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Rare sugars are the class of monosaccharide and their derivatives that hasrare presence in nature. In this class there are about 50 kinds of raresugar and one of them is D-Allose. Recent characterization of physiologicalfunction of these rare sugars revealed that D-Allose has a novel inhibitoryeffect on production of reactive oxygen species (ROS) on ischemic nervecells. We also found that D-Allose has a novel inhibitory effect on cellproliferation in HeLa (uterine cervical cancer), OVCAR-3 (ovarian cancer)and Daudi (Burkitt lymphoma) cells. Further, we analyzed these cancer cellstreated with D-Allose using a proteomic approach to identify the proteinsthat may play the important physiological role in apoptosis of cancer cells.Although we detect no change on Coomassie-stained SDS-PAGE of whole celllysates in the presence or absence of D-Allose, we found somepost-translational changes by Western blotting with anti-phosphorylationantibody. Since the various growth factor receptors in cell are controlledby phosphorylation, this may well work with as a single factor or in acombination to have inhibitory effect for the production of active ROS andmay control the cell division cycle that may result in apoptosis of cancercells. The further characterization of various candidate proteins is underway at molecular level by a proteomic approach. [Jpn J Physiol 54 Suppl:S84 (2004)]

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Liver sinusoidal endothelial cells (ECs) of the nonembolized hepatic lobe are stretched continuously followed by the increase in blood flow after portal vein embolization. Previously, we showed that ECs secret IL-6, a trigger of liver regeneration, via NF-κB pathway when they were subjected to continuous stretch on an elastic silicone chamber. In this study, we examined the mechanism of NF-κB activation in response to uni-axial continuous stretch in ECs. 1) Continuous stretch-induced NF-κB activation was inhibited by the application ofα5β1 integrin inhibitory peptide: GRGDNP, phospholipase C inhibitor: U73122, PKC inhibitor: H7 or the depletion of intra- and extracellular Ca2+ pools by both EGTA and thapsigargin (TG), but not inhibited by the application of a blocker for stretch-activeted channels: Gadolinium (Gd3+), the depletion of extracellular Ca2+ by EGTA or the depletion of intracellular Ca2+ stores by TG. 2) PKC kinase activity was inhibited by the application of GRGDNP or U73122 and the deplation of intra- and extracellular Ca2+ pools by both EGTA and TG. 3) Iincrease in intracellular Ca2+ concentration in response to continuous stretch was observed in the presence of EGTA or TG, but not observed in the presence of both EGTA and TG. These results indicate that NF-κB activation following IL-6 secration in response to continuous stretch is dependent on outside-in signaling at integrins followed by Ca2+-dependent PLC/PKC signaling pathway. [Jpn J Physiol 54 Suppl:S84 (2004)]

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In order to examine the dynamic properties of osteoclast activity, we observed their motile events under the cultured condition by using video-enhanced DIC microscope system. Osteoclasts were isolated from the long bones of the rabbit, and placed on coverslips coated with calcium phosphate. The cells were incubated in the chamber on the DIC microscope at 35 degrees without CO2 supplement, and observed sequentially every 3 s for 60 min. Osteoclasts showed filopodial and lamellipodial movements on the calcium phosphate coated glass. Surprisingly, a part of filopodia and lamellipodia fragmented and transported the substrate of calcium phosphate. Occasionally, vacuoles were formed in the central area of the cell body. These results suggest that the osteoclast destructs the bone mechanically by filopodial and lamellipodial activity in the peripheral area. [Jpn J Physiol 54 Suppl:S84 (2004)]

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It has been conceived that NaCl absorption in the gastrointestinal tract is mediated by coupled Na⁺/H⁺ and Cl⁻/HCO₃⁻ exchangers. NHE3 is a major Na⁺/H⁺ exchanger contributing to NaCl absorption; however, the molecular identity of the Cl⁻/HCO₃⁻ exchanger involved in NaCl absorption is uncertain. Recent studies have suggested that Down Çúregulated in adenoma (DRA, also called Slc26a3) is a major Cl⁻/HCO₃⁻ exchanger in the colon. Since multiple isoforms of the Cl⁻/HCO₃⁻ exchanger are co-expressed in an intact colonic cell, complicating the functional analysis of an individual isoform, we used heterologous transfection of DRA into cell lines. We generated a construct encoding the DRA tagged with the hemagglutinin (HA) epitope and transiently transfected the cells with this construct. The expression of the HA-tagged DRA by western blotting was verified. Two immunoreactive bands were detected in the transfected cells: a major protein of 120 kDa, probably glycosylated DRA, and a smaller band of 90 kDa, which is probably un-glycosylated DRA. This assumption was validated by treatment of the cells with an N-glycosylation inhibitor resulting in the disappearance of the higher molecular band. The subcellular localization of DRA was assessed by immunofluorescence experiments; DRA was found almost exclusively in the surface membrane. Since DRA expression was not stable and disappeared over weeks, we are now trying to establish the cells stably expressing DRA by using inducible gene expression systems. [Jpn J Physiol 54 Suppl:S84 (2004)]

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In 2002, we demonstrated in this meeting that allograft-induced cytotoxic T lymphocytes (CTLs) were highly cytotoxic against allografted lymphoid cells, P815 mastocytoma cells, and fibroblasts but were inactive toward skin, KLN205 squamous carcinoma cells, and Meth A fibrosarcoma cells. In the present study, we examined the role of perforin and Fas ligand (FasL), both of which are known as the major effector molecules of CTL, in the rejection of CTL-susceptible P815 (H-2^d) cells from gld (a mutation of FasL), perforin knockout (KO), perforin/FasL double-deficient (dd) or control C57BL/6 (H-2^b) mice. When P815 cells were i.p. transplanted into gld, perforin KO, dd or control mice, they acutely rejected the allograft with similar time courses after transplantation; and host cells infiltrating into the rejection site exhibited similar cytotoxic activities against the allografted cells in a 12-h assay, whereas the cytotoxic activities of perforin KO or dd mice were less than10% of those from control mice in a 4-h assay, suggesting an important role of perforin in the early phase of cytotoxic activity. In addition, the cytotoxic activities of dd mice suggested the involvement of adhesive molecules in the cytotoxic mechanism. These results taken together indicate that most of in vivo CTL activity against allografted P815 cells appeared to be dependent on a third mechanism, one other than perforin- and FasL-based pathways. [Jpn J Physiol 54 Suppl:S85 (2004)]

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We have reported that flavonoids stimulate Cl- secretion by activating Cl- channel and/or Na+/K+/2Cl- cotransporter depending upon their structures. Our previous study showed that the flavones such as quercetin, luteolin and apigenin stimulate Cl- secretion by activating the bumetanide-sensitive Na+/K+/2Cl- cotransporter and that these flavones suppress the cAMP-stimulated Cl- secretion without affecting apical Cl- conductances in renal epithelial A6 cells. Myricetin, a flavone, also stimulated the Cl- secretion by activating the Na+/K+/2Cl- cotransporter likely quercetin, luteolin and apigenin. In this study, we found that myricetin enhanced the Cl- secretion by increasing the apical Cl- conductance under the cAMP-stimulated condition. However, under the basal condition, myricetin stimulated the Cl- secretion without any increases in the apical Cl- conductance. These results suggest that myricetin stimulates only the cAMP-activated Cl- channel, but not the unactivated Cl- channel at the apical membrane. Supported by JSPS 15659052 (YM), 15590189 (NN). [Jpn J Physiol 54 Suppl:S85 (2004)]

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Ambroxol is often used as an expectorant in various lung diseases. However, it is unclear how ambroxol acts on bronchial epithelial cells. To clarify the action of ambroxol, we studied the effect of ambroxol on the ion transport in human Calu-3 cells, a human submucosal serous cell line, measuring the transepithelial short circuit current across monolayers of Calu-3 cells. Ambroxol of 100 micro-M applied to the both apical and basolateral sides diminished the terbutaline (a beta2-adrenergic agonist)-stimulated chloride secretion without any decreases in the apical chloride channel conductance. On the other hand, ambroxol had no effects on the chloride secretion or the apical chloride channel conductance under the basal (unstimulated) condition. Ambroxol had no major action on the epithelial Na+ channel (ENaC) and its-mediated Na+ absorption. These results indicate that: 1) ambroxol acts on the cAMP-stimulated Na+/K+/2Cl- cotransporter in Calu-3 cells, and 2) ambroxol has more powerful action than the adrenergic agonist action on the cAMP-stimulated Na+/K+/2Cl- cotransporter, leading the chloride secretion to a moderately stimulated level from a hyper-stimulated level. Supported by JSPS 15659052 (YM), 15590189 (NN), 15790120 (HM), MHLW 14C-5. [Jpn J Physiol 54 Suppl:S85 (2004)]

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Nasal cavity and paranasal sinus have various functions. However, there is little information about ion transport characteristics in these upper air way epithelia. The purpose of the present study is to evaluate the phenotype of ion transport in human nasal polyp (NP) and paranasal sinus mucosa (PSM) with or without bronchial asthma. NP and PSM tissues were obtained by endoscopic sinus surgery. Epithelial cells prepared from NP and PSM tissues were seeded on collagen coated dishes and cultured in bronchial epithelial growth media. Then, the cells were reseeded on culture inserts, and cultured in air liquid surface media. After culturing about 2 weeks, we mounted the cells into a modified Ussing chamber to characterize ion transport properties by measuring the short circuit current (ISC). The basal ISC of NP was mostly composed of the benzamil-sensitive Na+ absorption which was larger than that of PSN. This suggests that NP behaves as a Na+ absorbing tissue. The comparative study of the ion transport properties in NP with or without bronchial asthma showed that the benzamil-sensitive Isc in NP with bronchial asthma was much lower than that in NP without bronchial asthma. This result means that bronchial asthma makes the human nasal epithelium more secretory by inhibiting the Na+ absorption. Supported by JSPS 15659052 (YM), 15590189 (NN), 15790120 (HM). [Jpn J Physiol 54 Suppl:S86 (2004)]

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Mutations in presenilin 1 and 2 (PS1 and PS2) account for the majority of early-onset familial Alzheimer's disease (FAD). However, trafficking and interaction of PSs with other proteins are poorly understood. We have previously shown that PSs bound to syntaxin 5 (Syx5), which is a t-SNARE (target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) that is involved in ER-Golgi vesicular transport. By using co-immunoprecipitation and pull down studies, we further examined the interaction of PS with Syx isoforms expressed in HeLa, COS7 and NG108-15 cells. We found that ER-type isoform of Syx5 bound to holoproteins of PS1 and PS2. In contrast, Syx1A, 2, 3 and 4 that are located at the plasma membrane did not show specific binding to PS. In addition, Syx6, 7, 8 that localized in post-TGN (trans-Golgi-Network) did not bind to PS holoproteins. The FAD-linked PS1 variant (PS1deltaE9), which lacks the cytoplasmic loop that contains the endoproteolytic cleavage site, showed markedly decreased binding to Syx5. Interaction domains in PS were mapped to the cytoplasmic loop region and transmembrane domain (TMD). The interaction domains in Syx5 were mapped to the TMD and the cytoplasmic region that is distinct from SNARE motif. Taken together, these results indicate that Syx5 isoforms specifically bind to full-length PSs at ER and cis-Golgi compartment via interaction of their cytoplasmic and transmembrane regions. These results suggest that Syx5 isoforms may play a role in trafficking PSs from the ER to the Golgi in the early secretory pathway. [Jpn J Physiol 54 Suppl:S86 (2004)]

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D-serine is an endogenous modulator of N-methyl-D-aspartate (NMDA)/glutamate receptors. L-serine, the precursor of D-serine, is also essential for survival and development of neurons including cerebellar Purkinje neurons. We have recently identified a high affinity D- and L-serine transporter Asc-1 from mouse and human brain. In this study, we isolated a cDNA for the rat orthologue of Asc-1, and generated two kinds of polyclonal antibodies against Asc-1 based on the amino acid sequence of rat Asc-1. Using these antibodies, we performed immunohistochemical analysis to determine the localization of Asc-1 in rat brains. Asc-1 immunoreactivity was detected in neurons, including cerebellar Purkinje neurons and pyramidal neurons in the hippocampus and the neocortical layers III and V. It was clearly localized in dendrites as well as somata. Asc-1 immunoreactivity was also detected on neurons in other regions such as the cerebellar nuclei. The localization of Asc-1 in brain suggests the significant contribution of Asc-1 to amino acid mobilization in brains including the synaptic clearance of D-serine and the neuronal uptake of L-serine. [Jpn J Physiol 54 Suppl:S86 (2004)]

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<Introduction>Slc26 family and AE family are distinct structurally, but both mediate anion exchange at the plasma membrane. The purpose of this study is to identify, in small intestinal enterocytes, anion exchangers which belong to Slc26 family and AE family by intracellular pH (pH_i) measurements and by using RT-PCR.<Method> Top parts of villus were cut from mouse ileum, and loaded with BCECF-AM. Fluorescent measurement was performed on the stage of an inverted microscope. The samples were illuminated alternately at 440 and 500 nm, and the fluoresence was measured at 520-570 nm through interference filter. The pH_i was calibrated by the nigericin-high K⁺ method.<Result and Discussion> In HCO₃⁻/CO₂ buffer enterocytes were alkalized by removal of Cl⁻ and recovered by restoration of Cl⁻, whereas in HCO₃⁻/CO₂-free buffer the alkalization induced by Cl⁻ removal was not observed. This indicated that enterocytes had an activity of Cl⁻/HCO₃⁻ exchange. The pH_i recovery from alkalization induced by Cl⁻ removal was observed by addition of Br⁻, I⁻, F⁻, NO₃⁻, or SO₄^2-. The pH_i recovery by all attempted anions were smaller than that by Cl⁻. In addition, SO₄^2- did not recovered pH_i until baseline while others did. These results suggested that there were more than two functionally distinct Cl⁻/HCO₃⁻ exchangers in mouse ileal enterocytes, one accepting SO₄^2- in place of Cl⁻ (thus, probably a member of Slc26 family) and the other not. In additional experiments, we confirmed the expression of Slc26a3, Slc26a6 and AE2 in mouse ileal enterocyte by RT-PCR analysis. [Jpn J Physiol 54 Suppl:S86 (2004)]

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To determine the sugar binding site of the Na⁺/glucose cotransporter type1 (SGLT1), substrate specificity of a chimera transporter which was made from rabbit SGLT1 and Xenopus SGLT1-like protein (SGLT1L) was examined. The order of the substrate specificity of SGLT1 is D-glucose:=D-galactose:=α-methyl-D-glucoside (αMDG)>>myo-inositol while that of Xenopus SGLT1L is myo-inositol>D-glucose>D-galactose:=αMDG. αMDG is a specific substrate for SGLT. A chimera transporter was made by substituting the extracellular loop between the transmembrane domain (TM)12 and TM13 of Xenopus SGLT1L with the same portion of rabbit SGLT1. Then the chimera was expressed in the Xenopus oocyte membrane and its transport activity was measured by the two-microelectrode voltage-clamp method. The order of the substrate specificity of the chimera was myo-inositol:=D-glucose>D-galactose:=αMG showing that by the substitution the affinity of the chimera with D-glucose increased but the affinities with myo-inositol, D-galactose and αMG changed little when compared to the native Xenopus SGLT1L. As a result, the substrate specificity of the chimera transporter was similar to neither　SGLT1 nor Xenopus SGLT1L. Therefore, the extracellular loop between TM12 and TM13 is probably a part of the sugar binding site of SGLT1. [Jpn J Physiol 54 Suppl:S87 (2004)]

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The interlobular duct segments of guinea-pig pancreas secrete HCO₃⁻ into a HCO₃⁻-rich (125 mM) luminal fluid following secretin stimulation. Under these conditions intracellular HCO₃⁻ is ~20 mM and intracellular potential is ~-60 mV indicating a luminally directed electrochemical gradient for HCO₃⁻. To examine whether luminal HCO₃⁻ transport is mediated by an anion conductance, we measured changes in intracellular pH (pH_i) when the cells were de- or hyper-polarized by manipulation of extracellular K⁺ ([K⁺]_B). The isolated ducts were superfused with HCO₃⁻/CO₂-free solution and luminally perfused with 125 mM HCO₃⁻ and 24 mM Cl⁻. pH_i was measured with BCECF. When [K⁺]_B was raised from 5 to 70 mM, pH_i in the unstimulated ducts changed slightly. In the presence of dbcAMP, the depolarization caused a large increase in pH_i from 6.83 ± 0.11 to 7.32 ± 0.09 (mean ± SEM, n = 4, p<0.01). When [K⁺]_B was reduced from 5 to 1 mM, pH_i decreased by 0.11 ± 0.01 (p<0.05). Under Cl⁻-free conditions, when [K⁺]_B was reduced from 5 to 1 mM and then raised to 70 mM in the presence of dbcAMP, pH_i decreased from 7.15 ± 0.06 (n = 4) to 7.06 ± 0.07 and then increased to 7.54 ± 0.16 (p<0.05). In summary, de- and hyper-polarization caused changes in pH_i that most probably reflected the influx and efflux of HCO₃⁻ across the luminal membrane. These HCO₃⁻ movements were not dependent on the presence of Cl⁻ and may be attributed to the presence of a significant HCO₃⁻ conductance at the luminal membrane. [Jpn J Physiol 54 Suppl:S87 (2004)]

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The urate/anion exchanger URAT1 regulates blood urate level and is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region as identified by both yeast two-hybrid and in vitro binding assays. In addition, the first, second, and fourth PDZ domains within PDZK1 associate with the URAT1 C-terminal. Co-immunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. When transfected into HEK293 cells with pDsRed2-C1/PDZK1 and pEGFP-C2/ wild-type URAT1, PDZK1 colocalized with URAT1 on the surface membrane. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-folds), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the Vmax of urate transport via URAT1, although it was not associated with the high URAT1 protein level in crude membrane fractions from URAT1-expressing HEK293 cells prepared with or without PDZK1 transfection. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the membrane of renal proximal tubules. [Jpn J Physiol 54 Suppl:S87 (2004)]

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Although adenosine triphosphate (ATP) released from epithelia is an important autocrine and paracrine signaling, it is difficult to study due to its sensitiveness to mechanical stress. We have examined the cultured human bronchial epithelial cells (16HBE) to determine whether changes in Cl⁻ concentration or osmolality or shaking stimulate the cell to release ATP by using the luciferin-luciferase luminometric assay. Upon hypotonicity (205 mosmol kgH₂O⁻¹), the rate of ATP release increased from 0.036 ± 0.034 to 3.1 ± 0.6 pmol min⁻¹ at 6 min and decreased. The peak value of ATP release is estimated to be 6.2 x 10⁴ ATP molecules s⁻¹ per cell. Forskolin (10 μM) increased the hypotonicity-induced ATP release (P < 0.005), which was abolished (P < 0.001) by pretreatment with BAPTA-AM (25 μM), a membrane permeable Ca²⁺ chelator. Interestingly, lowering Ca²⁺ in the extracellular solution stimulated ATP released during mechanical perturbation, such as hypotonicity and gentle shaking. On the other hand, neither low Cl⁻ (75 mM) nor hypertonicity (500 mosmol kgH₂O⁻¹) could significantly increase the ATP release. Further, either forskolin or ionomycin failed to stimulate ATP release under the isotonic condition. In conclusion, a biphasic increase in ATP release and its low rate at the peak support the hypothesis that ATP is released through a non-conducting pathway model including exocytosis. Intracellular Ca²⁺ may play a key role for hypotonicity-induced ATP release. [Jpn J Physiol 54 Suppl:S87 (2004)]

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We have previously shown in rat brain that ABCA2, which belongs to the A subclass of ATP-binding cassette (ABC) transporter superfamily, is specifically expressed in the cytoplasm of myelin-forming oligodendrocytes in white and grey matter but not in non-myelin-forming, GFAP-positive astrocytes or CD11b-positive microglia. We also reported that ABCA2 is expressed in rat peripheral nerve, but the cell types of ABCA2-positive were not clarified. In addition, it is not known if ABCA2 is expressed in human nerve. Here we demonstrate that ABCA2 is expressed in human facial nerve cells that are either strongly positive or only weakly positive for the Ca²⁺-binding protein S100β. The former S100β-positive cells are Schwann cells. The latter ABCA2-positive/S100β-weakly positive cells may correspond to precursor satellite cells of rat peripheral nerve, which, as we reported previously, are round shaped and strongly positive for ABCA2 but only weakly positive for CNP. Cells that are S100β-positive but negative for ABCA2 also are present in human facial nerve. Since ABCA2 is not expressed in non-myelin-forming cells in brain, these cells may well be non-myelin-forming Schwann cells. These results suggest that ABCA2 might be used as a marker to differentiate non-myelin-forming and myelin-forming glial cells both in brain and peripheral nervous system. [Jpn J Physiol 54 Suppl:S88 (2004)]

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Aberrant HCO₃⁻ transport is a hallmark of cystic fibrosis (CF) and is associated with aberrant Cl⁻-dependent HCO₃⁻ transport by the cystic fibrosis transmembrane conductance regulator (CFTR). In a previous work, we showed that the bulk of CFTR activated Cl⁻-dependent HCO₃⁻ transport occurs as a coupled Cl⁻/HCO₃⁻ exchange and identitied the members of SLC26 anion exchangers as proteins activated by CFTR(EMBO J 2002, 21:5662). In the present study when co-expressed in HEK293 cells, we found that the first and the second half of the CFTR with Regulatory(R) domain [TM1+NBD1+R, R+TM2+NBD2] activated Down-regulated in adenoma (DRA; SLC26A3) anion exchanger, a gene responsible for a congenital chloride diarrhea. In contrast, DRA activated CFTR whole cell chloride current by ~3 fold although the amount of the expressed CFTR in the plasma membrane remained unchanged. DRA increased CFTR Po (open probability) and NPo by ~2.5 fold and 5 fold, respectively. In addition, a novel SLC26 transporter domain, DRA STAS domain interacts with CFTR and markedly increased the Po and NPo of CFTR by ~3 fold and 7 fold, respectively. Significantly, disease-associated mutant DRA I668ins and its STAS domain (DRA STAS I668ins) inhibited the CFTR chloride current. Taken together, these findings provide a molecular mechanism for CFTR-dependent HCO₃⁻ transport. Mutual regulatory interactions of CFTR and the member of SLC26 transporter may explain the aberrant HCO₃⁻ transport observed in CF. [Jpn J Physiol 54 Suppl:S88 (2004)]

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Intercellular canaliculi (IC) is a primary mixing reservoir for transcellularly and paracellularly secreted saliva whose composition is dependent on the degree of elevation of cytosolic Ca²⁺ and of cytosolic cyclic AMP concentrations caused by various secretagogues. In rat submandibular gland (SMG), appearance of exocytosis on IC reflected the amount of secreted mucin (Murakami 2002). Segawa (2002) showed in the parotid gland (PG) that, while at first transcellular fluid secretion predominates subsequently there is an increase of paracellular fluid transport through the tight junctions. By contrast, vigorous secretory stimulation at higher concentration of carbachol (1μM CCh) and isoproterenol (1μM ISP) caused enormous deformation of IC and decrease in ATP (Murakami 2002). The present morphological observation was carried out by HR-SEM using a modified osmium maceration method (Riva 1994) on specimens treated with CCh and/or ISP. Mild secretory stimulation revealed that exocytosis did not occur simultaneously even along the same intercellular canaliculus. Higher dose did not change the spatial distribution but increased the frequency of exocytosis. The temporal frequency of exocytosis along intercellular canaliculi was dose-dependent. These findings lead to conclude that under low level of secretory stimulation exocytosis does not show a dose-dependent change, but that its spatial and temporal frequency changes dose-dependently. [Jpn J Physiol 54 Suppl:S88 (2004)]

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The cytoplasmic calcium ion concentration is lower than that of extracellular space or intracellular calcium storage such as endoplasmic reticulum (ER) or mitochondria. The calcium influx from the extracellular space or the calcium release from the intracellular storage result in the change of this balance and this change is quite important for variable cellular functions.The initial step of protein exocytosis is the vesicular transport of protein from the ER to the Golgi apparatus. How can cytoplasmic calcium concentration change influence on the protein transport between ER and the Golgi? Ionomycin is a well known reagent which can release calcium from intracellular storage or can enhance the calcium influx from the extracellular space. When the protein transport kinetics is compared between ionomycin plus or minus conditions, it has been revealed that the protein transport between ER and the Golgi was inhibited in the presence of ionomycin. When the transport kinetics was examined with thapsigargin, which can release the calcium from the ER, we could not observe any inhibitory effect. These results indicate that the calcium influx from the outside of the cell may inhibit the protein transport between ER and the Golgi. [Jpn J Physiol 54 Suppl:S88 (2004)]

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We have reported phosphorylations of the basolateral membrane (BLM) proteins by membrane-bound A-kinase (PKA) by effect of cAMP in rat parotid gland (PG) acinar cells (Kurihara K et al. Am. J. Physiol. 279, C1516-1527, 2000). In the present study, we examined the distribution and function of PKA/A-kinase anchoring protein (AKAP) complex, in order to elucidate the regulation of Na/K-ATPase by phosphorylation among three major salivary glands, i.e., submandibular gland (SMG), sublingual gland (SLG), and PG. Both AKAP-150 protein and its mRNA were clearly detected in the PG, but not detected in SMG and SLG. Membrane-bound form of the RII regulatory subunit, an index for the amount of membrane-anchored PKA holo-enzyme, was substantially present in membranes from PG, but less detectable in those from SMG and SLG. AKAP-150 in PG acinar cells was co-immunoprecipitated with RII regulatory subunit by an anti-RII antiserum, suggesting that AKAP-150 is indeed functional in anchoring RII molecules in vivo. Thus, the AKAP-150/PKA complex is expressed and functions specifically in PG. Furthermore, Na/K-ATPase in the PG was phosphorylated by membrane-anchored endogenous PKA triggered by the addition of cAMP, but those in the SMG and SLG membranes were not. The regulation of Na/K-ATPase by phosphorylation associated to AKAP-anchored PKA is a characteristic rather specific for PG among the three major salivary glands. [Jpn J Physiol 54 Suppl:S89 (2004)]

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The A subclass of the ATP-binding cassette (ABC) transporter superfamily has a structural feature that distinguishes it from other ABC transporters, and is proposed to be involved in transmembrane transport of endogenous lipids. Here we cloned the full-length cDNA of a novel ABC transporter ABCA14, which belongs to the A subclass. Mouse ABCA14 is a protein of 1,733 amino acid residues, and is most homologous to the mouse ABCA3 and sea urchin ABCA (suABCA), with amino acid identity of 55.2% and 35.4%, respectively. RNA blot and quantitative real-time PCR analyses showed that ABCA14 mRNA is exclusively expressed in the testis. Examination of testis by in situ hybridization determined that ABCA14 mRNA is expressed in germ cells including spermatogonia and spermatocytes in the seminiferous tubule. Immunoblot analysis using a specific antibody showed that ABCA14 is a protein of 200kDa, and immunohistochemical analysis showed that the protein is detected in the region of anterior head of sperm and elongated spermatids. Since the removal of cholesterol from the sperm plasma membrane is thought to be critical during capacitation, ABCA14 may play a pivotal role in the efflux of endogenous cholesterol during sperm capacitation. [Jpn J Physiol 54 Suppl:S89 (2004)]

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Ca²⁺ entry pathways in rat submandibular acinar cellsYoshida, H. and Nakahari, T.Department of Physiology, Osaka Medical College, 2-7 Daigaku-cho, Takatsuki 569-8686, JapanIn rat submandibular acinar cells, 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 extracellular fluid, which is triggered by empty stores and is the so-called "store-operated Ca²⁺ entry". Store-operated Ca²⁺ entry was stimulated using 4 microM thapsigargin, sarco(end)plasmic reticulum Ca²⁺ ATPase inhibitor, in Ca²⁺-free solution (no added Ca²⁺ + 1mM EGTA) followed by superfusion with control (Ca²⁺-containing) solution. This protocol resulted in a [Ca²⁺]_i increase, which was inhibited in the presence of 100 microM Gd³⁺, 100microM La³⁺ but was unaffected by addition of 1 microM Gd³⁺ or 10 microM H-89, PKA inhibitor. A restoration from a high K+ solution(Ca²⁺-containing) to control solution also evoked an [Ca²⁺]_i increase during thapsigargin stimulation, and an [Ca²⁺]_i increase following reintroduction of Ca²⁺ during thapsigargin stimulation was completely inhibited by addition of 1 microM Gd³⁺ but was partially affected by addition of 10 microM H-89 . Thus, one effect of the reintroduction of Ca²⁺ is likely to be a hyperpolarization voltage. In conclusion, rat submandibular acinar cells have at least two Ca²⁺ entry pathways; one is activated by the store-depletion, and the other is by cAMP and hyperpolarization. [Jpn J Physiol 54 Suppl:S89 (2004)]

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Prostaglandin E₂ (PGE₂) and substance P (SP) have been reported to induce massive Cl⁻ secretion in the guinea pig distal colon. It has been reported that this synergistic action of PGE₂ and SP is occurred by crosstalk between cAMP and Ca²⁺ second messenger systems via each receptor in epithelial cells. In the present study, we pharmacologically and histologically identified the PGE₂ receptors (EP_1-4 and DP receptors) for the synergistic Cl⁻ secretion in the guinea pig distal colon. In pharmacological experiments, mucosa-submucosal preparations of guinea pig distal colon were mounted in Ussing chamber, and short-circuit current (I_sc) was measured as an index of ion transport across the epithelium. All the experiments were performed in the presence of tetrodotoxin and piroxicam to remove the influence of neurons and endogenous prostaglandin production. As a result, EP₂ and DP receptor subtypes were predominant receptors for synergistic action of PGE₂ and SP. In immunohistochemistry, EP₂ receptor-immunoreactivity (-IR) was observed in almost all epithelial membrane in the guinea pig distal colon. EP₄ receptor-IR was observed in some epithelial cells. On the other hand, EP₁ and EP₃ receptor-IRs were not observed in mucosa. These results of immunohistochemistry are consistent with the pharmacological results. Therefore, the present study indicates that EP₂ receptors on epithelial membrane contribute to the synergistic Cl⁻ secretion by PGE₂ and SP. [Jpn J Physiol 54 Suppl:S89 (2004)]

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Background: Hypertension impairs endothelium-dependent vasodilation with aging. Aim: To examine age-related participation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in spontaneously hypertensive rats (SHRs). Methods: Coronary small arteries (100μm<) and arterioles (<100μm) in in vivo beating hearts of 6-, 12- and 28-week-old SHRs (n=41) and age-matched Wistar-Kyoto rats (WKYs) (n=38) were observed by our intravital microscope with CCD camera under cyclooxygenase blockade. Coronary vascular responses to acetylcholine (ACh) were examined under two conditions; ACh and ACh plus L-NAME. Results: Vasodilation of both arterioles and small arteries was maintained by 12 weeks in SHRs, but NO-mediated dilation decreased by 26% in arterioles and 34% in small arteries at 28 weeks compared to control. In 28 week-old-SHRs, contribution of NO-mediated vasodilation decreased by 24% in arterioles and 45% in small arteries, while contribution of EDHF-mediated dilation decreased by 27% in arterioles and 56% in small arteries. In WKYs, EDHF-mediated dilation decreased in small arteries by 31% of control, although ACh-dependent dilation was maintained by 28 weeks. Conclusion: Hypertension with aging at 28 weeks induces endothelial impairment of both NO and EDHF in small arteries as well as arterioles, while in normal rats, aging at 28 weeks attenuates EDHF-mediated dilation only in small arteries. [Jpn J Physiol 54 Suppl:S90 (2004)]

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Background:Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that plays a key role in the regulation of gene expression in response to hypoxia. We investigated that HIF-1α gene transduced endothelial progenitor cell (EPCs) established enhanced vascular development in severe ischemic hindlimb animals when transplanted in vivo. In this time, we focused the mechanism of over-expression of HIF-1α gene activity in vitro.Methods & Results:We isolated peripheral blood mononuclear cells from human volunteers, cultured through days 7, transducted adenovirus encoding HIF-1α gene (Tf/HIF-1α) and β-galactosidase transducted EPCs (Tf/β) gene as control to ex vivo expanded EPCs with previous condition, and evaluated proliferative activity, and migration activity, adhesion molecule, and apoptosis assay. 1) Tf/HIF-1α EPCs augmented cell proliferative activity and enhanced cell migration compared to Tf/β. 2) Tf/HIF-1α supported significantly more rolling and adhesion of EPCs compared to Tf/β on IL-1β activated HUVEC monolayer using dilution flow chamber. 3) Tf/HIF-1α EPC apoptosis was markedly reduced more than controls after gene transfer.Conclusion:This strategy will clear more some mechanism of hypoxic inducible pathway and will provide the potential application of gene therapy for vascular regeneration. [Jpn J Physiol 54 Suppl:S90 (2004)]

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A 3-minute ischemia/reperfusion caused expressions of angiogenic factors, bFGF and VEGF in 2 hours, PCNA in 24 to 96 hours in cardiac tissues, followed by an increase in capillaries in one month. X-ray (20 gray) exposure of rats also increased bFGF and VEGF, and capillary density in one month. A simple i.v. injection of bFGF or VEGF failed to cause any capillary increase. Thus, microdamages in cardiac tissues seemed necessary for the rapid increase in capillaries. Trials were made to elucidate the underlying mechanisms. In SHRSP the hypertrophy of cardiomyocytes exceeded the growth of capillaries, resulting in a low capillary density. Treatments of SHRSP using ACEI and/or angiotensin receptor blocker suppressed the hypertrophy in cardiomyocytes and increased the capillary density. Administration of clenbuterol to normal rats caused hypertrophy of cardiomyocytes accompanied with the reduction in the capillary density. The hypertrophy probably reduced the interstitial space among cardiomyocytes and suppressed the capillary proliferation. Expressions in bFGF and VEGF, and an increase in capillary density were found in 20 week-old OLETF rats (a model rat for type2 diabetes). However, an increase was found in PAI-1 expression in 40 to 60 week-old OLETF without any significant increase in capillary density. The expression of PAI-1 probably reduced the destruction rate in the extracellular matrix by plasmin, resulting in a retardation in the capillary proliferation. These data suggested that matrix destruction is required for the capillary proliferation and that the destruction may be accelerated by the 3 minute transient ischemia in cardiac tissues. [Jpn J Physiol 54 Suppl:S90 (2004)]

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The decrease in blood flow and the increase in sympathetic nerve activity to kidney are occurred during exercise. The feedforward neural drive from higher brain (central command) adjusts autonomic nervous system and evokes cardiovascular responses. We investigated whether central command increases renal sympathetic nerve activity (RSNA) and decreases renal blood flow (RBF), using mid-collicular decerebrate and unanesthetized rats (n=17). Rats were instrumented with laser flowmeter probe in a kidney (ALF21, Advance) (n=10), an electrode on renal nerves (n=7), and a catheter in carotid artery (all rats). Central command was evoked for 30 s by electrical stimulation of midbrain in paralyzed rats. The electrical stimulation was applied to the area where it evoked behavior (20–60 μA current for 1 ms, 60 Hz). The stimulation significantly increased arterial pressure (peak change from baseline at 60 μA current: +60±6 mmHg, mean±S.E.) and RSNA (+210±30%), and decreased RBF (–34±6%). Those changes were greater as current intensity of the stimulation was greater. The decrease in RBF evoked by central command was abolished after cutting renal nerves. Our findings suggest that central command decreases RBF by the increase in RSNA. [Jpn J Physiol 54 Suppl:S91 (2004)]

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Venous stasis caused by immobility is an important risk factor for deep vein thrombosis (DVT) following surgery, in bed-ridden medical patients, and in long distance travelers. To prevent venous stasis and then DVT, intermittent pneumatic compression (IPC) and graduated compression stocking (GCS) are widely applied. IPC is thought to prevent venous stasis by intermittent external compression, and GCS is thought to shift superficial venous flow to the deep vein by continuous external compression. On the other hand, muscle contraction itself can increase blood flow by autoregulation and can prevent venous stasis by muscle pump. Recently, we proposed a new device which is constructed by GCS and electrical muscle stimulation of calf muscles (active stocking, patent 2003-206335). In the present study, effectiveness of ICP, GCS, and active stocking on deep venous hemodynamic was examined using color Doppler imaging in 7 healthy volunteers (male=5, female=2, age: 18-37 years). Subjects have no history of DVT, peripheral arterial disease, or venous insufficiency. IPC and GCS increased femoral venous blood flow by 13 ± 6, and 17 ± 7%, respectively. On the other hand, active stocking elicited a significantly larger increase in femoral venous blood flow (56 ± 10%) compared to IPC and CGS. Thus, active stocking might have potential for preventing DVT by improving deep venous hemodynamic. To test this, clinical trials are needed. [Jpn J Physiol 54 Suppl:S91 (2004)]

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To clarify the capillary hemodynamics in terms of longitudinal and transverse orientation of capillary, we measured red blood cell velocity (VRBC) for these two orientations of capillary in soleus, i.e. straight and tortuous capillaries, and capillary in gastrocnemius, i.e. straight and anastomotic capillaries, at rest in anesthetized rat with our newly developed pencil-probe charge-coupled device (CCD) videomicroscope. Five to ten regions of interest (ROIs) were chosen from the surface of these muscles. The rats were assigned to one of two groups: control, or hindlimb suspension for 2 weeks (HS). As for control, the longitudinal and transverse VRBC for soleus was significantly lower than that for gastrocnemius. HS significantly decreased longitudinal VRBC for gastrocnemius and significantly increased transverse VRBC for soleus. Coefficient of variation (CV = SD/mean), which reflects heterogeneity of VRBC among ROIs, of VRBC was not significantly changed. The frequency distribution of Vrbc was described by a gamma distribution except for that of longitudinal Vrbc in atrophying soleus. These results indicate that adaptive microvascular hemodynamics in atrophying muscle is different between soleus and gastrocnemius muscle. [Jpn J Physiol 54 Suppl:S91 (2004)]

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Human fingers are an excellent organ in sensory function as well as motor function. We hypothesized that blood vessels of the fingers are dynamically regulated by sympathetic outflow during concentrated finger perception. To identify this hypothesis, we measured finger skin blood flow (FSBF), heart rate (HR), and mean arterial blood pressure (MAP) during finger reading performed under the blind condition. A two-dimensional image of FSBF (palmar digital artery of the second-third fingers; scan area, 5X5cm; spatial resolution, 1mm²) was measured using a laser Doppler perfusion imaging equipment. Finger skin vascular conductance was calculated from a ratio of the blood flow and MAP. The subjects were instructed to read a flat plate with raised letters (diameter, 3mm; height, 1mm) for 15 sec by the forefinger, and to touch the flat plate without any raised letters. HR and MAP slightly increased during finger reading but unchanged during touching the blank plate. FSBF was decreased during touching the plate with the raised letters, whereas the blood flow did not be affected by touching the flat plate. The mean decrease in FSBF was 19±3% during finger reading, while contralateral FSBF unchanged. The changes in finger skin vascular conductance followed the changes in FSBF, because the changes in MAP were negligible. These results indicated that blood flow in the fingers is decreased during finger reading. We considered that sympathetic outflow to the hand may contribute to this decrease in blood flow. [Jpn J Physiol 54 Suppl:S91 (2004)]

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Space sickness is believed to be related to the cephalad fluid shift and elevated intracranial pressure (ICP) during early microgravity exposure. Recently, we have reported elevation of ICP and cerebral perfusion pressure (CPP) although cerebral flow velocity (CFV) did not change during microgravity induced by free drop. We hypothesized that resistance of cerebral blood vessel increased to counteract against increase in cerebral blood flow (CBF) due to cephalad fluid shift. To test the hypothesis, we measured abdominal aortic pressure (AAP), carotid arterial and venous pressure (CAP and CVP, respectively), CBF (n = 10), and muscle blood flow (MBF) (n = 6) during microgravity induced by parabolic flight. The rats were set at horizontal prone (Flat) and 30-degree head-up whole body tilting (HUT) positions. With HUT, CVP increased by 1.9 mmHg during microgravity, compared to 1G condition. CAP raised by 15 mmHg although AAP rose by only 8 mmHg right after induction of microgravity. Similarly, in spite of increase in MBF up to 160%, only 115% increase in CBF was observed. With Flat position, no significant change was shown during microgravity. These results indicate that cephalad fluid shift due to acute microgravity exposure cause sudden increase in peripheral blood flow but change in CBF is counteracted by cerebral hemodynamic autoregulation. [Jpn J Physiol 54 Suppl:S92 (2004)]

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The effect of shear stress on nitric oxide (NO)-mediated attenuation of anaphylactic vasoconstriction was studied using an isolated guinea pig liver perfused under constant flow or pressure. The flow constant resulted in increased shear stress during constriction; the perfusion pressure steady prevented changes in shear stress. Using the double occlusion technique to estimate the hepatic sinusoidal pressure (Pdo), pre- (Rpre) and post-sinusoidal (Rpost) resistances were calculated. In either constant flow or pressure perfusion, anaphylaxis contracted predominantly pre-sinusoids over post-sinusoids, a finding consistent with the previous study (Ruan et al. Am J Physiol Regul Integr Comp Physiol, in press). When shear stress was allowed to rise, the presinusoidal constriction, but not post-sinusoidal constriction, was significantly potentiated after NO synthase blockade using N^G-nitro-L-arginine methyl ester (L-NAME, 100 μM). When shear stress was held constant by maintaining constant perfusion pressure, L-NAME potentiated both pre- and post-sinusoidal constriction in a similar magnitude. The data suggest that hepatic anaphylaxis generates NO in a shear stress independent manner, resulting in dilatation of both pre- and post-sinusoidal vessels. In contrast, NO was preferentially generated shear stress dependently at the vigorously contracted pre-sinusoids and dilate predominantly that pre-sinusoids in isolated guinea pig liver with anaphylactic venoconstriction. [Jpn J Physiol 54 Suppl:S92 (2004)]

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During lymphatic transport, lymph fluid travels through several regional lymph nodes. There are, however, few information regarding lymphatic flow pathway of substances and lymphocytes within the lymph nodes. We have established suitable preparations to visualize the lymphatic flow pathway within lymph nodes by using fluorescent video-microscope system (Jpn. J. Physiol., 52: S67, 2002). In the present study, we investigated quantitatively the lymphatic flow pathway of fluorescence substances and lymphocytes within the lymph nodes. The iliac lymph nodes were isolated from Wistar rats and their afferent and efferent lymph vessels were cannulated in an organ chamber. Lymphocytes were collected from the mesenteric lymph nodes and then provided to label for visualization with CSFE. The isolated lymph nodes were selectively circulated with physiological salt solution through the afferent and efferent lymph vessels in the absence or presence of fluorescence substances (FITC-dextran; 77KDa and fluoresbrite carboxylate microsphere; 1μm) and CSFE-labeled lymphocytes. The lymphatic flow pathway within the lymph nodes was obtained by using fluorescence video-microscopy. The FITC-dextran passed through the lymph nodes about 120sec, while the microspheres and lymphocytes were captured within the lymph nodes. These results suggest that there are heterogeneities in the molecular size-dependent lymphatic flow pathways within the lymph nodes. [Jpn J Physiol 54 Suppl:S92 (2004)]

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We have developed a quantitative filtration apparatus to evaluate erythrocyte deformability, using a precision metal filter (nickel mesh) which we developed. The filtration system consists of a test unit (a vertical glass tube equipped with a nickel mesh at the bottom end), a negative pressure supply unit, and a measurement control and data analysis unit. The vertical-tube is connected to an air tank or reservoir through electromagnetic valves inside the negative pressure supply unit. The test material is sucked into the filter through the action of decreasing negative pressure. The device automatically gives a liquid level (height)-time relation and a pressure-flow rate relation, similar to gravity-based filtration. Unlike the gravity-based system, the experimental performance of this system is very good and reproducible. Nowadays, the physiologic significance and clinical benefit of nickel mesh filtration is evident. Thus, we believe that our new, highly quantitative and convenient apparatus will contribute to the evaluation of erythrocyte deformability for biorheology and related fields of medical science. [Jpn J Physiol 54 Suppl:S92 (2004)]

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Objective: To visualize and analyze spatiotemporal dynamics of individual living monocyte during transendothelial migration (TEM). Methods and Results: We developed a new experimental system to observe TEM with following two improvements. (1) Ultra thin collagen gel layer (30-50 μm thick) constructed under endothelium for 3D observation with high magnification. (2) Appropriate fluorescent labeling of living monocytes and endothelial cells to keep highest cell activity. Individual monocyte behaved quite diversely. About 70% of adhered monocytes directionally crawled to intercellular junction. Time from adhesion to start of invasion was 8.6±5.4 min (n=61 monocytes). However, remaining 30% did not transmigrate. Some of them could not find the invasion spot despite actively crawled on endothelium. Time from start to finish of invasion was 6.3±3.2 min (n=53 monocytes). About 20% of once invading monocytes hesitated transmigration, and returned onto endothelial surface. Conclusions: Using our new system, we visualized and quantitatively analyzed for the first time detailed spatiotemporal, 3D dynamics of individual living monocyte during TEM. Complete transmigration of individual monocyte could be achieved only when both vitality of the monocyte and the local condition of endothelium constructively couple together to proceed TEM. [Jpn J Physiol 54 Suppl:S93 (2004)]

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