研究者詳細 - 成瀬　恵治

論文 - 成瀬　恵治

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Involvement of SA channels in orienting response of cultured endothelial cells to cyclic stretch 査読

K Naruse, T Yamada, M Sokabe

AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY 274 ( 5 ) H1532 - H1538 1998年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER PHYSIOLOGICAL SOC

The present work was designed to elucidate the involvement of Ca2+-permeable stretch-activated (SA) channels in the orienting response of endothelial cells to uniaxial cyclic stretch. Endothelial cells from human umbilical vein were cultured on an elastic silicons membrane and subjected to uniaxial cyclic stretch (120% in length, 1 Hz). The cells started to change their morphology 15 min after the onset of stretch, and >90% of the cells oriented perpendicularly to the stretch axis after 2 h. Associated with the orienting response, cell elongation proceeded with a slower rate. Both of the orientating and elongating responses were largely inhibited by the removal of external Ca2+ or by Gd3+, a potent blocker for the SA channel, but not by nifedipine. Intracellular Ca2+ concentration ([Ca2+](i)) transiently increased in response to uniaxial stretch, and the basal [Ca2+](i) gradually increased during cyclic stretch. This Ca2+ response was inhibited by the removal of extracellular Ca2+ or by the addition of Gd3+. These results suggest that stretch-dependent Ca2+ influx through SA channels is essential in the stretch-dependent cell orientation and elongation.

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Up-regulation of COX2 expression by uni-axial cyclic stretch in human lung fibroblast cells 査読

T Kato, N Ishiguro, H Iwata, T Kojima, T Ito, K Naruse

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 244 ( 3 ) 615 - 619 1998年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

The effect of uni-axial cyclic mechanical stretch on the expression of cyclooxygenases (COX) was investigated in a human lung fibroblast cell line (TIG-1). In response to uni-axial cyclic stretch, the level of COX2 mRNA significantly increased and peaked at 3 h (9.09 +/- 3.82-fold, mean +/- standard error, n = 6, compared with that at 1 h). The level of the expression of COX2 protein peaked at 6 h, whereas the level of COX1 protein was not significantly changed. The involvement of stretch-activated (SA) channel was investigated in the stretch-induced COX2 production. The application of Gd3+, a blocker for SA channel, or the removal of extracellular Ca2+ inhibited the production of COX2 mRNA without any effect on the production of COX1 or GAPDH mRNA. These data strongly suggest that COX2 expression is up-regulated by uni-axial cyclic stretch via the activation of SA channel in human lung fibroblasts. (C) 1998 Academic Press.

DOI： 10.1006/bbrc.1998.8335

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Both focal adhesion kinase and c-ras are required for the enhanced matrix metalloproteinase 9 secretion by fibronectin in ovarian cancer cells 査読

K Shibata, F Kikkawa, A Nawa, AA Thant, K Naruse, S Mizutani, M Hamaguchi

CANCER RESEARCH 58 ( 5 ) 900 - 903 1998年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER ASSOC CANCER RESEARCH

Cell adhesion to the extracellular matrix appears to trigger a cascade of intracellular signalings. We have shown previously that treatment of ovarian cancer cells with peritoneal conditioned medium or purified fibronectin (FN) activated matrix metalloproteinase 9 secretion and, thereby, cancer cell invasion, By use of antisense oligonucleotides to focal adhesion kinase (FAK) and a dominant-negative mutant of ras (S17Nras), we found that both FAK and c-Ras were required for the activation of matrix metalloproteinase 9 secretion by FN. In addition, both antisense oligonucleotides to FAR and S17Nras inhibited mitogen-activated protein kinase activation hy FN treatment, suggesting the involvement of mitogen-activated protein kinase in the FN-dependent signaling.

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Bystander tumoricidal effect and gap junctional communication in lung cancer cell lines 査読

K Imaizumi, Y Hasegawa, T Kawabe, N Emi, H Saito, K Naruse, K Shimokata

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY 18 ( 2 ) 205 - 212 1998年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER LUNG ASSOC

Tumor cells expressing the herpes simplex virus-thymidine kinase (HSV-tk) gene become sensitive to ganciclovir (GCV), and the phenomenon by which tumor cells surrounding the HSV-tk expressing cells also become sensitive to CCV is known as the "bystander effect." The purpose of this study was to investigate the bystander effect in human lung-cancer cell lines, and the role of gap-junctional intercellular communication as the mechanism responsible for it. Gap-junctional intercellular communication was measured both with a dye-transfer assay involving single-cell microinjection of Lucifer Yellow and with a PKH26/calcein-AM double-dye-transfer assay. Significant bystander tumoricidal effect was observed in lung-cancer cell lines when cultured cells contained only 10% HSV-tk expressing cells. This was also observed to occur with cell lines of different origin or from different species. Although gap-junctional intercellular communication characterized by rapid transfer of Lucifer Yellow was not observed, we did detect gap-junctional communication marked by the slow transfer of calcein-AM in lung-cancer cell lines. However, neither an inhibitor (I-octanol) nor an enhancer (all trans-retinoic acid [ATRA]) of gap-junctional communication affected the extent of the bystander effect. These findings suggest that low levels of gap-junctional communication may be efficient for producing the bystander effect in lung-cancer cells, or chat other mechanisms may underlie this effect. Although gap-junctional communication may play an important role in generating the bystander effect in tumor cells expressing the HSV-tk gene, further knowledge of the mechanism of this effect may help improve the treatment of lung cancer with an HSV-tk system.

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Up-regulation of integrin beta(3) expression by cyclic stretch in human umbilical endothelial cells 査読

N Suzuki, K Naruse, Y Asano, T Okamoto, N Nishikimi, T Sakurai, Y Nimura, M Sokabe

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 239 ( 2 ) 372 - 376 1997年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS

The effect of uni-axial cyclic mechanical stretch on the expression of the adhesion protein integrin was investigated. Human umbilical endothelial cells (HUVECs) cultured an fibronectin coated silicon membranes were subjected to uni-axial cyclic stretch, The level of expression of integrin beta(3) mRNA was found to be increased and peaked at 4 hours in response to cyclic stretch using a semiquantitative RT-PCR method. The increased level of the integrin mRNA from stretched HUVECs remained higher than that ii om non-stretched controls. The amount of integrin beta(3) also increased and peaked at 12 hr, Immuno-fluorescent microscopy revealed that the amount of integrin beta(3) adhesions increased in stretched HUVECs compared with that in non-stretched HUVECs. These results suggest that uni-axial cyclic stretch up-regulates the expression of integrin beta(3). This increase in integrin beta(3), may enhance the adhesiveness to the substratum and contribute to the protection of HUVECs against being peeled off from the vessel wall. (C) 1997 Academic Press.

DOI： 10.1006/bbrc.1997.7364

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Mechanotransduction and intracellular signaling mechanisms of stretch-induced remodeling in endothelial cells 査読

M Sokabe, K Naruse, S Sai, T Yamada, K Kawakami, M Inoue, K Murase, M Miyazu

HEART AND VESSELS ( Suppl 12 ) 191 - 193 1997年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER

We investigated the signaling mechanism of stretch-induced cell remodeling in human umbilical vein endothelial cells (HUVECs). Freshly dissociated HUVECs were cultured on an elastic silicon membrane and subjected to uniaxial cyclic stretch (20% in length, 1Hz). The cells started to change their morphology as early as 15 min after stretch onset, and most cells eventually aligned perpendicularly to the stretch axis within Ih. This remodeling was dependent on the increase in intracellular calcium concentration ([Ca2+](i)) via a Ca2+-permeable stretch-activated (SA) channel. During the process of remodeling, extensive rearrangement of stress fibers and focal adhesions was observed, which may be close to the final step in the intracellular signaling cascade. This event was [Ca2+](i)-dependent, suggesting the existence of a Ca2+-dependent intermediate cascade that links [Ca2+](i) to the rearrangement of cytoskeletons and focal adhesions. We found that some proteins, including pp125(FAK) (focal adhesion kinase) and paxillin, were tyrosine phosphorylated during cyclic stretch in a Ca2+-dependent manner. Inhibition of this tyrosine phosphorylation prohibited the stretch-dependent rearrangement of cytoskeletons and focal adhesions as well as the remodeling. Finally the tyrosine kinase src, which could phosphorylate pp125(FAK), was found to be activated in a [Ca2+](i)-dependent way during stretch. All of the above molecular events were consistently Ca2+-dependent, which led us to propose the signaling cascade: SA channel activation --> [Ca2+](i) increase --> src activation --> protein tyrosine phosphorylation --> rearrangement of cytoskeletons and focal adhesions --> cell remodeling.

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Mechanosensitive ion channels: single channel vs. whole cell activities 査読

Sokabe M, Naruse K, Nunogaki K

Prog Cell Res 6 139 - 149 1997年

Effects of repetitive stretch stimulation on neonatal rat cardiocytes in vitro 査読

Kada K, Yasui K, Naruse K, Toyama J

Environmental Medicine 40 ( 1 ) 69 - 72 1996年

Inhibitory action of repeated stretch stimulation on apoptosis in neonatal rat cardiocytes 査読

Yasui K, Shimano H, Kada K, Naruse K, Toyama J

Environmental Medicine 40 ( 2 ) 175 - 177 1996年

PLATEAU PATTERN OF AFFERENT DISCHARGE RATE FROM FROG-MUSCLE SPINDLES 査読

M SOKABE, K NUNOGAKI, K NARUSE, H SOGA, N FUJITSUKA, A YOSHIMURA, F ITO

JOURNAL OF NEUROPHYSIOLOGY 70 ( 1 ) 275 - 283 1993年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER PHYSIOLOGICAL SOC

1. A characteristic plateau pattern was observed in the rate of afferent discharges during ramp-and-hold stretch of spindles isolated from semitendinosus muscles of frogs. The plateau pattern was more frequent in summer frogs (84%) than winter frogs (11%).
2. The plateau pattern consisted of a discharge rate around 120 imp/s at the end of dynamic stretch, followed by second and third steps of plateau rates around 60 and 40 imp/s, respectively. The intervals of impulses in lower steps were approximately n times those of the top step.
3. The plateau pattern was not sensitive to cutting extracapsular myelinated branches or lowering temperature from 23 to 12-degrees-C. However, the number of the plateau was reduced in both cases.
4. Application of depolarizing current to the sensory terminal abolished the plateau pattern. In contrast, in spindles that did not show a plateau pattern, hyperpolarizing current induced such a pattern.
5. Calcium channel blockers and protein kinase C inhibitors abolished the plateau pattern. The plateau pattern could be established in quiescent spindles by drugs eliciting Ca2+ entry, raising cytosolic-free Ca2+, and activating protein kinase C.
6. The most striking aspect of the present study is the stability in the discharge rate at each step of the plateau, irrespective of different experimental conditions. This suggests that the spindle sensory terminal possesses a stable intrinsic rhythm generator in excitation, of which maximum frequency is 120 imp/s. The generator seems to be triggered by stretch stimulus and to be regulated by cytoplasmic Ca2+ and protein kinase C.

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INVOLVEMENT OF STRETCH-ACTIVATED ION CHANNELS IN CA-2+ MOBILIZATION TO MECHANICAL STRETCH IN ENDOTHELIAL-CELLS 査読

K NARUSE, M SOKABE

AMERICAN JOURNAL OF PHYSIOLOGY 264 ( 4 ) C1037 - C1044 1993年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER PHYSIOLOGICAL SOC

Endothelial cells are subjected to shear stresses by blood flow, normal stresses by blood pressure, and stretch by vessel expansion. These forces are known to induce secretions of several vasoactive substances probably via internal calcium mobilization (R. F. Furchgott. Circ. Res. 53: 557-573, 1983; M. J. Peach, A. L. Loeb, H. A. Singer, and J. Saye. Hypertension Dallas 7, Suppl. I: I-94-1-100, 1985.). Here we report that stretching cellular membranes increased intracellular Ca2+ concentration ([Ca2+]i) in human umbilical endothelial cells cultured on silicon membranes. Upon application of a stretch pulse (3-s duration), [Ca2+]i increased rapidly and decayed slowly. The following results suggest that this increase arises from Ca2+ entry through stretch-activated (SA) channels: 1) the Ca2+ response disappeared when extracellular Ca2+ was removed; 2) gadolinium (Gd3+), a blocker for cation-selective SA channels, blocked the response but nifedipine did not; and 3) externally applied Mn2+, which is known to permeate mechanosensitive channels but not Ca2+ channels, entered the intracellular space immediately after an application of mechanical stretch. The increase in [Ca2+]i was found to consist of at least two components: an initial fast component and a delayed slower component. Ryanodine inhibited the slow component. It is suggested that stretching the membrane primarily induced extracellular Ca2+ entry through SA channels followed by Ca2+ releases from intracellular Ca2+ stores.

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MECHANICS OF PATCH CLAMPED AND INTACT CELL-MEMBRANES IN RELATION TO SA CHANNEL ACTIVATION 査読

M SOKABE, K NUNOGAKI, K NARUSE, H SOGA

JAPANESE JOURNAL OF PHYSIOLOGY 43 ( Suppl 1 ) S197 - S204 1993年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：CENTER ACADEMIC PUBL JAPAN

Stretch activated (SA) channels are believed to be activated by tension in the membrane generated by membrane stretch. However, very few studies have been made on the quantitative estimation of the tension during membrane stretch. Here we present a method to evaluate the tension both in patch clamped and intact cell-membranes. The tension in patch clamped membranes was calculated from Laplace's law by knowing transmembrane pressure and the radius of patch-curvature. We also provide a simpler version for calculating the tension from the pressure and pipette radius. The tension in intact cell membranes was calculated from Hook's law based on the measurement of changes in cell surface area. The estimated tension required for activating SA channels in both types of membranes was found to be comparable suggesting that,the SA channel acts as a physiological mechanotransducer in intact cells.

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DIFFERENTIAL RESPONSES OF CA-ACTIVATED K-CHANNELS TO BRADYKININ IN SENSORY NEURONS AND F-11 CELLS 査読

K NARUSE, DS MCGEHEE, GS OXFORD

AMERICAN JOURNAL OF PHYSIOLOGY 262 ( 2 ) C453 - C460 1992年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER PHYSIOLOGICAL SOC

The nonapeptide bradykinin (BK) excites a subset of dorsal root ganglion (DRG) neurons with putative nociceptive functions by stimulating an inward cation current. In addition, BK stimulates various intracellular signaling pathways including an elevation of intra-cellular Ca2+. In a DRG neuron x neuroblastoma hybrid cell (F-11), BK stimulates similar increases in intracellular [Ca2+] and inward current but also elicits a large transient outward current through Ca2+-activated K channels. We have investigated the mechanisms underlying differential expression of outward current responses in the two cell types at the single channel level. Although K(Ca) channel activity appears in inside-out patches from both cells exposed to Ca2+, BK applied to the extrapatch membrane of cell-attached patches activates K(Ca) channels in F-11 but not DRG neurons. Whereas single K(Ca) channels are quantitatively similar in terms of conductance, voltage-dependence, and sensitivity to tetraethylammonium, they differ in sensitivity to intracellular Ca2+. Channel activation in both cells requires at least four Ca2+ ions, but half-maximal activation occurs at slightly higher [Ca2+] for DRG neurons. The shift in the Ca2+ dose-response curve combined with the steep [Ca2+] dependence of channel open probability makes it less likely that a BK-induced rise in internal [Ca2+] induced will trigger a transient outward current and resultant hyperpolarization in a DRG neuron.

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ELECTROPHYSIOLOGICAL ANALYSIS OF STRUCTURAL ASPECTS OF VOLTAGE-DEPENDENT SR K+ CHANNEL 査読

M SOKABE, M KASAI, K NOMURA, K NARUSE

COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-PHARMACOLOGY TOXICOLOGY & ENDOCRINOLOGY 98 ( 1 ) 23 - 30 1991年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD

This article presents a brief review on the electrophysiological analysis of the structural aspects of the voltage-dependent SR (sarcoplasmic reticulum) K+ channel. In the first half, early attempts to determine the physical dimensions of the ion conducting mechanism such as the mouth, narrow tunnel, or ion selective filter of the channel, are reviewed. The depicted cartoon of the SR K+ channel, as an extremely short, busy district with a big mouth on each side, is quite similar to the recently-obtained reconstructed structural image of the acetylcholine receptor channel. In the latter half, we introduce our recent attempts to draw a physical image of the gating mechanism of the SR K+ channel. We examined, for example, the location of the gate and the voltage sensor, and the relationship between them. It is suggested that the gate and the sensor are connected tightly and that the sensor would be exposed to the surface of the lumen side of SR when the gate opens. Finally, the issue of substates in SR K+ channel is discussed. It is implied that the substate-conductances reflect a partial occlusion of the pore by an intermediate-open gate.

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CLASSIFICATION OF THE INTRAFUSAL MUSCLE-FIBERS IN THE FROG-MUSCLE SPINDLE - HISTOCHEMICAL AND IMMUNOFLUORESCENT STUDIES 査読

A YOSHIMURA, N FUJITSUKA, M SOKABE, K NARUSE, K NOMURA, FH DIWAN, F ITO

JOURNAL OF ANATOMY 172 89 - 101 1990年10月

AMINOGLYCOSIDE BLOCKADE OF CA2+-ACTIVATED K+ CHANNEL FROM RAT-BRAIN SYNAPTOSOMAL MEMBRANES INCORPORATED INTO PLANAR BILAYERS 査読

K NOMURA, K NARUSE, K WATANABE, M SOKABE

JOURNAL OF MEMBRANE BIOLOGY 115 ( 3 ) 241 - 251 1990年5月

Effects of ions and drugs on the responses of sensory axon terminals of decapsulated frog muscle spindles 査読

Fumio Ito, Masahiro Sokabe, Kazushi Nomura, Keiji Naruse, Noriaki Fujitsuka, Atsushi Yoshimura

Neuroscience Research Supplements 12 ( C ) S15 - S26 1990年

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