研究者詳細 - 成瀬　恵治

論文 - 成瀬　恵治

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Protamine augments stretch induced calcium increase in vascular endothelium 査読

K Murase, K Naruse, A Kimura, K Okumura, T Hayakawa, M Sokabe

BRITISH JOURNAL OF PHARMACOLOGY 134 ( 7 ) 1403 - 1410 2001年12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：WILEY

1 Human umbilical vein endothelial cells cultured on a transparent silicone chamber were subjected to a short stretch pulse (ca. 1 s, 5-25% stretch) of their substrate and following increases in intracellular Ca2+ concentration ([Ca2+](i)) were measured by fluorescence intensity ratiometry using fura-2.
2 In response to mechanical stretch, the cells in HEPES buffered saline exhibited a Ca2+ transient in a dose dependent way. The response was completely dependent on external Ca2+ and inhibited by gadolinium (Gd3+), suggesting that it was mediated by the activation of a stretch activated cation channel (SACatC).
3 Interestingly, the stretch induced Ca2+ transient was significantly augmented in the presence of basic polypeptide, protamine. This augmented Ca2+ response was inhibited neither by Gd3+ nor by the deprivation of external Ca2+, indicating that the SACatC is not responsible for this phenomenon.
4 In contrast, this augmentation was inhibited by depletion of intracellular Ca2+ stores with thapsigargin or by the pretreatment with phospholipase inhibitors such as U73122 and manoalide.
5 These results suggest the presence of a metabotropic mechanoreceptor distinct from the SACatC in vascular endothelium. This augmented [Ca2+](i) increase may contribute to the vasodilating response induced by protamine during heparin neutralization in cardiac surgery.

DOI： 10.1038/sj.bjp.0704386

Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation 査読

JG Wang, M Miyazu, E Matsushita, M Sokabe, K Naruse

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 288 ( 2 ) 356 - 361 2001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 ELSEVIER SCIENCE

We investigated the role of tyrosine phosphorylation of FAK in the stretch-induced MAPKs (extracellular signal-regulated kinase (ERK), p38MAPK) activation in mutant FAK-transfected fibroblasts. In response to uniaxial cyclic stretch (1 Hz, 120% in length), the levels of tyrosine phosphorylation of the Tyr-397 and Tyr-925 of FAK in control cells increased and peaked at 5 min (2.75 +/- 0.51, n = 3), and 20 min (2.98 +/- 0.58, n = 3), respectively, and the activities of MAPKs increased and peaked at approximately 10 min. On the other hand, in the mutant FAK-transfected cells, the stretch-induced MAPKs activation was significantly inhibited. The stretch-induced activation of MAPKs was also significantly abolished by either treatment with Gd3+ or extracellular Ca2+ removal which may inhibit intracellular Ca2+ increase caused by the activation of cation selective (Ca2+-permeable) stretch activated (SACatC) channels. These results suggest that the stretch-induced tyrosine-phosphorylation of FAK via SACatC activation is critical for the stretch-induced MAPKs activation. (C) 2001 Academic Press.

DOI： 10.1006/bbrc.2001.5775

SA channel mediates superoxide production in HUVECs 査読

K Aikawa, N Nishikimi, T Sakurai, Y Nimura, M Sokabe, K Naruse

LIFE SCIENCES 69 ( 15 ) 1717 - 1724 2001年8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：PERGAMON-ELSEVIER SCIENCE LTD

Superoxide production in response to cyclic stretch (1 Hz, 20 % in length) was investigated in human umbilical vein endothelial cells (HUVECs). The basal production of superoxide without stretch increased gradually, while the production of superoxide with stretch increased significantly as compared to that without stretch and it became significant 80 min after the onset of cyclic stretch (P<0.05, n=8-14). The superoxide production increased in a stretch-dependent manner and became significant when stretch was more than 10 % (p<0.05, n= 11-16). To investigate the involvement of SA channel, we added Gd3+ or EGTA in the reaction solution and examined the stretch-induced superoxide production. In cells stretched in the presence of 20 muM Gd3+, the stretch-induced superoxide production was significantly inhibited (at 120 min, p<0.05, n=8-18). The cyclic stretch-induced superoxide production was also significantly inhibited by the removal of extracellular Ca2+ with 5 mM EGTA (at 120 min, p<0.05, n = 8-18). Neither the application of Gd3+ nor the removal of extracellular Ca2+ significantly changed the basal production of superoxide. These data suggest that the stretch-induced superoxide production increases in time- and stretch-dependent manner and that the stretch-induced superoxide production in HUVECs is regulated by Ca2+ influx through SA channels. (C) 2001 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S0024-3205(01)01256-5

Mechanical behavior in living cells consistent with the tensegrity model 査読

N Wang, K Naruse, D Stamenovic, JJ Fredberg, SM Mijailovich, IM Toric-Norrelykke, T Polte, R Mannix, DE Ingber

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 98 ( 14 ) 7765 - 7770 2001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：NATL ACAD SCIENCES

Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors, Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

DOI： 10.1073/pnas.141199598

Subcellular positioning of small molecules 査読

Takayama S, Ostuni E, LeDuc P, Naruse K, Ingber DE, Whitesides GM

Nature 411 ( 6841 ) 1016 - 1016 2001年6月

Stretch-induced morphological changes of human endothelial cells depend on the intracellular level of Ca2+ rather than of cAMP 査読

T Yamada, K Naruse, M Sokabe

LIFE SCIENCES 67 ( 21 ) 2605 - 2613 2000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：PERGAMON-ELSEVIER SCIENCE LTD

When exposed to a uni-axial cyclic stretch, cultured human umbilical vein endothelial cells (HUVECs) align and elongate perpendicular to the stretch axis. Previous studies showed that forskolin inhibited stretch-induced orientation of endothelial cells, suggesting that adenosine3:5-cyclic monophosphate (cAMP) plays an important role in the shape change. However, we have recently shown that stretch-induced shape changes in cultured HUVECs are due to increased [Ca2+],. In the present study, we examined the possible role of cAMP in stretch-induced shape changes in cultured HUVECs, Application of uni-axial cyclic stretch induced a gradual rise in cAMP reaching a peak level at 60 min after the onset of stretch. The adenylate cyclase activator, forskolin, increased the basal level of cAMP but inhibited the rise in [Ca2+](i) resulting in no cell shape changes. In contrast, N 6,2-dibutyryladenosine3:5-cyclic monophosphate (dbcAMP) enhanced the stretch-induced increase in cAMP and [Ca2+] and resulted in cell shape changes. On the other hand, 2'5'-dideoxyadenosine (DDA), an adenylate cyclase inhibitor, inhibited stretch-induced increases in cAMP and [Ca2+]i resulting in no cell shape changes. In summary, our data showed that cell shape changes were consistently dependent on [Ca2+](i) rather than cAMP levels. We conclude that the primary second messenger in the stretch-induced shape changes in HUVECs is intracellular Ca2+ rather than cAMP. (C) 2000 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S0024-3205(00)00844-4

Aldose reductase inhibition prevents glucose-induced apoptosis in cultured bovine retinal microvascular pericytes 査読

K Naruse, J Nakamura, Y Hamada, M Nakayama, S Chaya, T Komori, K Kato, Y Kasuya, K Miwa, K Naruse, N Hotta

EXPERIMENTAL EYE RESEARCH 71 ( 3 ) 309 - 315 2000年9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 LTD

The pathogenesis of pericyte loss, an initial deficit in the early stage of diabetic retinopathy, remains unclear. Polyol pathway hyperactivity has been implicated in the pathogenesis of diabetic retinopathy, and recent studies have suggested that apoptosis may be involved in pericyte loss. The present study was conducted to investigate whether high glucose induces apoptosis in cultured bovine retinal pericytes. The effect of an aldose reductase inhibitor. SNK-860, was also examined. After a 5 day incubation with various concentrations of glucose (5.5-40 mM) in the presence or absence of SNK-860, the cell viability and the percentages of dead cells were measured. and staining with the TUNEL method and Hoechst 33342, and DNA electrophoresis were performed. High glucose reduced the viability and increased the percentages of dead cells. TUNEL-positive cells were observed in pericytes under high glucose, but not in those under 5.5 mM glucose. In the staining of nuclei with Hoechst 33342, the percentage of apoptotic cells in total cells counted under high glucose was higher than that under 5.5 mM glucose. DNA electrophoresis of pericytes cultured with high glucose demonstrated a 'ladder pattern'. Hyperosmolarity also induced apoptosis in pericytes, but less than that by high glucose. SNK-860 inhibited the glucose-induced apoptosis in pericytes. These observations suggest that the pericyte loss in diabetic retinopathy involves an apoptotic process, and that the polyol pathway hyperactivity plays an important role in inducing apoptosis in pericytes by high glucose. (C) 2000 Academic Press.

DOI： 10.1006/exer.2000.0882

Erratum: 'Molecular identification of a eukaryotic, stretch-activated nonselective cation channel' (Science (Aug. 6, 1999) (882)) 査読

M. Kanzaki, M. Nagasawa, I. Kojima, C. Sato, K. Naruse, M. Sokabe, H. Iida

Science 288 ( 5470 ) 1347 2000年5月

Molecular identification of a eukaryotic, stretch-activated nonselective cation channel 査読

M Kanzaki, M Nagasawa, Kojima, I, C Sato, K Naruse, M Sokabe, H Iida

SCIENCE 285 ( 5429 ) 882 - 886 1999年8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 ADVANCEMENT SCIENCE

Calcium-permeable, stretch-activated nonselective cation (SA Cat) channels mediate cellular responses to mechanical stimuli. However, genes encoding such channels have not been identified in eukaryotes. The yeast MID1 gene product (Mid1) is required for calcium influx in the yeast Saccharomyces cerevisiae. Functional expression of Midi in Chinese hamster ovary cells conferred sensitivity to mechanical stress that resulted in increases in both calcium conductance and the concentration of cytosolic free calcium. These increases were dependent on the presence of extracellular calcium and were reduced by gadolinium, a blocker of SA Cat channels. Single-channel analyses with cell-attached patches revealed that Midi acts as a calcium-permeable, cation-selective stretch-activated channel with a conductance of 32 picosiemens at 150 millimolar cesium chloride in the pipette. Thus, Midi appears to be a eukaryotic, SA Cat channel.

DOI： 10.1126/science.285.5429.882

An aldose reductase inhibitor prevents the glucose-induced increase in PDGF-beta receptor in cultured rat aortic smooth muscle cells 査読

Y Kasuya, J Nakamura, Y Hamada, M Nakayama, H Sasaki, T Komori, S Chaya, G Watanabe, K Naruse, E Nakashima, K Kato, N Hotta

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 261 ( 3 ) 853 - 858 1999年8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

To examine the role of platelet-derived growth factor (PDGF) and the polyol pathway in the growth activity of smooth muscle cells (SMCs), [H-3]-thymidine incorporation, [I-125]-PDGF-BB binding and expression of PDGF-beta receptor protein were measured in rat aortic SMCs cultured with 5.5 or 20 mM glucose with or without anti-PDGF antibody or an aldose reductase inhibitor, epalrestat. SMCs cultured with 20 mM glucose demonstrated an accelerated thymidine incorporation compared with SMCs cultured with 5.5 mM glucose, which was prevented by anti-PDGF antibody. This acceleration of growth activity by 20 mM: glucose was accompanied by an increase in PDGF-BB binding, which was due to the increased number of PDGF-beta receptors and the overexpression of PDGF-beta receptor protein. Epalrestat prevented all these abnormalities. These observations suggest that polyol pathway hyperactivity plays an important role in the proliferation of SMCs which may be mediated through the accelerated expression of PDGF-beta receptor protein. (C) 1999 Academic Press.

Characterization of a newly found stretch-activated K-Ca,K-ATP channel in cultured chick ventricular myocytes 査読

T Kawakubo, K Naruse, T Matsubara, N Hotta, M Sokabe

AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY 276 ( 6 ) H1827 - H1838 1999年6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

With the use of the patch-clamp technique, five kinds of stretch-activated (SA) ion channels were identified on the basis of their single-channel conductances and ion selectivities in cultured chick ventricular myocytes. Because a high-conductance K+-selective channel predominated among these channels, we concentrated on characterizing its properties mostly using excised inside-out patches. With 145 mM KCl solution in the pipette and the bath, the channel had a conductance of 199.8 +/- 8.2 pS (n = 22). The ion selectivities among K+, Na+, Ca2+, and Cl- as estimated from their permeability ratios were P-Na/P-K = 0.03, P-Ca/P-K = 0.025, and P-Cl/P-K = 0.026. The probability of the channel being open (P-0) increased with the Ca2+ concentration in the bath ([Ca2+](b); dissociation constant Kd = 0.51 mu M at +30 mV) and membrane potential (voltage at half-maximal P-o, = 39.4 mV at 0.35 FIM [Ca2+](b)). The channel was blocked by gadolinium, tetraethylammonium, and charybdotoxin from the extracellular surface and, consequently, was identified as a Ca2+-activated K+ (K-Ca) channel type. The channel was also reversibly activated by ATP applied to the intracellular surface (K-d = 0.74 mM at 0.10 mu M [Ca2+](b) at + 30 mV). From these data taken together, we concluded that the channel is a new type of K-Ca channel that could be designated as an "SA K-Ca,K-ATP channel. "To our knowledge, this is the first report of K-Ca channel in heart cells.

Activation of pp60(src) is critical for stretch-induced orienting response in fibroblasts 査読

XR Sai, K Naruse, M Sokabe

JOURNAL OF CELL SCIENCE 112 ( 9 ) 1365 - 1373 1999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：COMPANY OF BIOLOGISTS LTD

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120-130 kDa) increased and peaked at 30 minutes, Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120-130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an antisense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1. than in 3Y1 fibroblasts, These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

Appropriately spaced nuclear localizing signals are necessary for efficient nuclear import of nonnuclear proteins 査読

HJW Borgeld, K Naruse, S Nishikawa, J Zhang, A Kikuchi, K Furukawa, K Yagi, M Tanaka

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 256 ( 2 ) 278 - 283 1999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

To deliver nonnuclear proteins into the nucleus, we have examined the locations and number of nuclear localizing signals by use of simian virus 40 large T-antigen (SV40Ta) and yeast enhanced green fluorescent protein (yEGFP) in Saccharomyces cerevisiae as a model system. When only one SV40Ta was added to either the N- or C-terminus of yEGFP, the fluorescence of yEGFP was detected in both the nucleus and the cytoplasm. When two SV40Ta signals were added, one to the N-terminus and one to the C-terminus of yEGFP (SV40Ta-yEGFP-SV40Ta), the fluorescence of yEGFP was localized in only the nucleus. When the presequence of cytochrome oxidase subunit IV (pCOXIV) was inserted between the SV40Ta and the N-terminus of yEGFP (SV40Ta-pCOXIV-yEGFP-SV40Ta) in this construct, the fluorescence was located in both the nucleus and the cytoplasm, suggesting that the increased distance between the two SV40Ta signals decreased the efficiency of transport into the nucleus. When an additional SV40Ta signal was inserted between pCOXIV and yEGFP (SV40Ta-pCOXIV-SV49Ta-yEGFP), the fluorescence was localized only in the nucleus, indicating that two SV40Ta signals spaced by pCOXIV of 28 amino acid residues forming an alpha-helix are potent in transporting yEGFP into the nucleus. These results indicate that two SV40Ta signals spaced appropriately are essential for the efficient transport of the nonnuclear protein into the nucleus. (C) 1999 Academic Press.

DOI： 10.1006/bbrc.1999.0269

Prostacyclin synthase gene transfer accelerates reendothelialization and inhibits neointimal formation in rat carotid arteries after balloon injury 査読

Y Numaguchi, K Naruse, M Harada, H Osanai, S Mokuno, K Murase, H Matsui, Y Toki, T Ito, K Okumura, T Hayakawa

ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY 19 ( 3 ) 727 - 733 1999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：LIPPINCOTT WILLIAMS & WILKINS

Prostacyclin (PGI(2)), a metabolite of arachidonic acid, has the vasoprotective effects of vasodilation, anti-platelet aggregation, and inhibition of smooth muscle cell proliferation. We hypothesized that an overexpression of endogenous PGI(2) may accelerate the recovery from endothelial damage and inhibit neointimal formation in the injured artery. To test this hypothesis, we investigated in vivo transfer of the PGI(2) synthase (PCS) gene into balloon-injured rat carotid arteries by a nonviral lipotransfection method. Seven days after transfection, a significant regeneration of endothelium was observed in the arteries transfected with a plasmid carrying the rat PCS gene (pCMV-PCS), but little regeneration was seen in those with the control plasmid carrying the lacZ gene (pCMV-lacZ) (percent luminal circumference lined by newly regenerated endothelium: 87.1+/-6.9% in pCMV-PCS-transfected vessels and 6.9+/-0.2% in pCMV-lacZ vessels, P<0.001). BrdU staining of arterial segments demonstrated a significantly lower incorporation in pCMV-PCS-transfected vessels (7.5+/-0.3% positive nuclei in vessel cells) than in pCMV-lacZ (50.7+/-9.6%, P<0.01). Moreover, 2 weeks after transfection, the PCS gene transfer resulted in a significant inhibition of neointimal formation (88% reduction in ratio of intima/media areas), whereas medial area was similar among the groups. Arterial segments transfected with pCMV-PCS produced significantly higher levels of 6-keto-PGF(1 alpha), the main metabolite of PGI(2), compared with the segments transfected with pCMV-lacZ (10.2+/-0.55 and 2.1+/-0.32 ng/mg tissue for pCMV-PCS and pCMV-placZ, P<0.001). In conclusion, this study demonstrated that an in vivo PCS gene transfer increased the production of PGI(2) and markedly inhibited neointimal formation with accelerated reendothelialization in rat carotid arteries after balloon injury.

Orientation change of cardiocytes induced by cyclic stretch stimulation: Time dependency and involvement of protein kinases 査読

K Kada, K Yasui, K Naruse, K Kamiya, Kodama, I, J Toyama

JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY 31 ( 1 ) 247 - 259 1999年1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 LTD- ELSEVIER SCIENCE LTD

Mechanical stress has been implicated as one of the growth regulators in the heart. We investigated the effect of cyclic stretch stimulation on morphology and orientation of cultured cardiocytes. Embryonic rat (17 days postcoital) cardiomyocytes cultured on silicone dishes were cyclically stretched to 120% in length at a frequency of 30 cycles/min. After 12 h, in the initial stage of cultivation, cardiocytes and intracellular myofibrils oriented parallel to the stretch direction. When the stretch stimulus was prolonged to 24-48 h, myofibrils that oriented perpendicular to the stretch direction emerged. Furthermore. when the cells were stretched only in the later stage (after 24 h of cultivation). both cells and myofibrils tended to orient perpendicular to the stretch direction. Next we examined the effects of chemical compounds on these phase related changes in myofibril orientation None of the drugs tested (H-7, HA-1004, staurosporine, herbimycin A, genistein, GdCl3, and EGTA) blocked the parallel orientation of myofibrils induced by the initial-stage stretch. By contrast, H-7, staurosporine, herbimycin A, and genistein did inhibit almost completely the perpendicular orientation of the myofibrils induced by the late-stage stretch, but HA-1004, GdCl3, or EGTA did not, Immunoblotting study using anti-phsophotyrosine antibody indicated that tyrosine phosphorylation of a protein of about 125 kDa was enhanced in a time-dependent manner by the late-stage stretch, but not by the initial-stage stretch. In conclusion: the alignment change induced by cyclic stretch depends on the stage of cultivation: with stretch in the initial stage (within 12 h), cells and myofibrils orient parallel to the stretch; with stretch in the later stage (after 24 h), they orient perpendicular to the stretch. The effect of stretch in the later stage is likely mediated by protein kinase C and tyrosine kinase pathways. (C) 1999 Academic Press.

DOI： 10.1006/jmcc.1998.0865

An aldose redutase inhibitor prevents the intimal thickening in coronary arteries of galactose-fed beagle dogs 査読

Y. Kasuya, M. Ito, J. Nakamura, Y. Hamada, M. Nakayama, S. Chaya, T. Komori, K. Naruse, E. Nakashima, K. Kato, N. Koh, N. Hotta

Diabetologia 42 ( 12 ) 1404 - 1409 1999年

記述言語：英語掲載種別：研究論文（学術雑誌）

Aims/hypothesis. Although increased polyol pathway activity has been implicated in the pathogenesis of diabetic microangiopathy, the relation with diabetic macroangiopathy remains unclear. Galactose feeding is known to stimulate the polyol pathway and to develop abnormalites similar to those in diabetic microangiopathy. Our study was conducted to investigate whether an activation of polyol pathway by long-term treatment with galactose produced morphological changes in coronary arteries of dogs and the effect of an aldose reductase inhibitor, epalrestat, was also studied. Methods. Dogs received either normal chow or chow containing 30% galactose with or without epalrestat given orally (20 or 50 mg · kg-1). After 44 months, morphometric analyses of coronary arteries were carried out and the galactitol contents in aortas were measured. Results. The ratio of areas of the intimal layer to those of the medial layer, an indicator of intimal thickening, was statistically significantly increased in galactose-fed dogs compared with control dogs. Galactose-fed dogs had a remarkable accumulation of galactitol in their aortas. These morphological and biochemical deficits were reduced by treatment with epalrestat. Conclusion/interpretation. This report morphologically shows diabetes-like macrovascular abnormalities in galactosaemic animals, suggesting that polvol pathway hyperactivity is closely related to the development of diabetic macroangiopathy, which could be prevented by aldose reductase inhibition.

DOI： 10.1007/s001250051310

Uni-axial cyclic stretch induces c-src activation and translocation in human endothelial cells via SA channel activation 査読

K Naruse, Sai, X, N Yokoyama, M Sokabe

FEBS LETTERS 441 ( 1 ) 111 - 115 1998年12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：WILEY

The kinase activity of c-src increased and peaked at 15 min after an application of uni-axial cyclic stretch in HUVECs followed by a translocation of c-src to Triton-insoluble fraction. Suppression of c-src by an antisense S-oligodeoxynucleotide inhibited the stretch-induced tyrosine phosphorylation and morphological changes. The stretch-induced increase in c-src activity was inhibited by FK506, a specific inhibitor for calcineurin, by Gd3+, a blocker for stretch activated channels, and by the extracellular Ca2+ depletion suggesting the involvement of SA channels. These results strongly suggest c-src plays an important role in the downstream of SA channel activation followed by the morphological changes. (C) 1998 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(98)01528-2

Peroxide production and apoptosis in cultured cells carrying mtDNA mutation causing encephalomyopathy 査読

J Zhang, M Yoneda, K Naruse, HJW Borgeld, JS Gong, S Obata, M Tanaka, K Yagi

BIOCHEMISTRY AND MOLECULAR BIOLOGY INTERNATIONAL 46 ( 1 ) 71 - 79 1998年9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 AUST

When cybrids with a point mutation, which locates in the tRNA(Leu(UUR)) gene of mtDNA and causes a mitochondrial encephalomyopathy (MELAS syndrome), were exposed to a high concentration of oxygen (95%), the peroxide production markedly increased by 6 h of oxygen exposure, whereas the peroxide production was similar among the cybrids under a normal concentration of oxygen. The peroxide production by oxygen exposure was enhanced particularly in cybrids with high proportions of the mutant mtDNA and low respiratory capacities. The appearance of apoptotic cells by oxygen exposure was high in cybrids with the impaired respiratory function due to the mutation. An antioxidant NAC successfully suppressed both the peroxide production and apoptosis. These results imply that the peroxide production plays an important role in inducing apoptosis in :ells carrying; the mtDNA mutation causing encephalomyopathy.

Pp125(FAK) is required for stretch dependent morphological response of endothelial cells 査読

K Naruse, T Yamada, XR Sai, M Hamaguchi, M Sokabe

ONCOGENE 17 ( 4 ) 455 - 463 1998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：NATURE PUBLISHING GROUP

In this study, critical signaling pathway required for the stretch induced morphological changes of human umbilical endothelial cells (HUVECs) was investigated. Uniaxial cyclic stretch (1 Hz, 20% in length) of the cells cultured on an elastic silicon membrane induced a gradual morphological change in the cells from a polygonal shape to an elongated spindle-like shape whose long axis was aligned perpendicular to the stretch axis, We found that protein tyrosine phosphorylation of cellular proteins increased and peaked at 20 min in response to cyclic stretch. Either treatment of cells with gadolinium (Gd3+), a potent blocker for stretch-activated channels, or removal of extracellular Ca2+ blocked the tyrosine phosphorylation of the proteins, suggesting that stretch-activated (SA) ion channels regulated stretch specific tyrosine phosphorylation, The major phosphorylated proteins had molecular masses of approximately 120-135 kDa, and 70 kDa, Immunoprecipitation experiments revealed that paxillin, focal adhesion kinase (pp125(FAK)) and pp130(CAS) were included in the 70 kDa and 120-135 kDa bands, respectively, The morphological change was inhibited by herbimycin A and genistein, inhibitors of tyrosine kinases, suggesting that tyrosine phosphorylation was required for the morphological change, In addition, the kinase activation of pp125(FAK) was observed in response to cyclic stretch, Moreover, suppression of pp125(FAK) expression by the antisense phosphorothioate oligodeoxynucleotides (S-ODN) in HUVECs resulted in inhibition of tyrosine phosphorylation of paxillin and the stretch-dependent morphological changes. These results suggest that an activation of tyrosine kinase(s) by an increase in intracellular Ca2+ and pp125(FAK) play a critical role in the unique morphological change specifically observed in endothelial cells subjected to uni-axial cyclic stretch.

DOI： 10.1038/sj.onc.1201950

Involvement of SA channels of cultured endothelial cells 査読

Keiji Naruse, Takako Yamada, Masahiro 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）

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 silicone 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 &gt
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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