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1.  GADD34 suppresses lipopolysaccharide-induced sepsis and tissue injury through the regulation of macrophage activation 
Cell Death & Disease  2016;7(5):e2219-.
Growth arrest and DNA damage inducible protein 34 (GADD34) is induced by various cellular stresses, such as DNA damage, endoplasmic reticulum stress, and amino-acid deprivation. Although the major roles of GADD34 are regulating ER stress responses and apoptosis, a recent study suggested that GADD34 is linked to innate immune responses. In this report, we investigated the roles of GADD34 in inflammatory responses against bacterial infection. To explore the effects of GADD34 on systemic inflammation in vivo, we employed a lipopolysaccharide (LPS)-induced murine sepsis model and assessed the lethality, serum cytokine levels, and tissue injury in the presence or absence of GADD34. We found that GADD34 deficiency increased the lethality and serum cytokine levels in LPS-induced sepsis. Moreover, GADD34 deficiency enhanced tissue destruction, cell death, and pro-inflammatory cytokine expression in LPS-induced acute liver injury. Pro-inflammatory cytokine production after LPS stimulation is regulated by the Toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway. In vitro experiments revealed that GADD34 suppressed pro-inflammatory cytokine production by macrophages through dephosphorylation of IKKβ. In conclusion, GADD34 attenuates LPS-induced sepsis and acute tissue injury through suppressing macrophage activation. Targeting this anti-inflammatory role of GADD34 may be a promising area for the development of therapeutic agents to regulate inflammatory disorders.
PMCID: PMC4917654  PMID: 27171261
2.  Multiple roles of the PGE2-EP receptor signal in vascular permeability 
British Journal of Pharmacology  2014;171(21):4879-4889.
Background and Purpose
PGE2 is a major prostanoid that regulates inflammation by stimulating EP1–4 receptors. However, how PGE2 induces an initial inflammatory response to vascular hyper-permeability remains unknown. Here we investigated the role of the PGE2-EP receptor signal in modulating vascular permeability both in vivo and in vitro.
Experimental Approach
We used a modified Miles assay and intravital microscopy to examine vascular permeability in vivo. Endothelial barrier property was assessed by measuring transendothelial electrical resistance (TER) in vitro.
Key Results
Local administration of PGE2, an EP2 or EP4 receptor agonist into FVB/NJcl mouse ear skin caused vascular leakage, indicated by dye extravasation. Intravital microscopy and laser Doppler blood-flow imaging revealed that these treatments dilated peripheral vessels and increased local blood flow. Pretreatment with the vasoconstrictor phenylephrine inhibited the PGE2-induced blood flow increase and vascular leakage. In contrast to the EP2 and EP4 receptor agonists, administration of an EP3 receptor agonist suppressed vascular leakage without altering vascular diameter or blood flow. In isolated HUVECs, the EP3 receptor agonist elevated TER and blocked thrombin-induced dextran passage. Inhibiting PKA restored the hypo-permeability induced by the EP3 receptor agonist.
Conclusions and Implications
Activation of the PGE2-EP2 or -EP4 receptor signal induces vasodilatation in mural cells, resulting in increased local blood flow and hyper-permeability. In contrast, activation of the PGE2-EP3 receptor signal induces a cAMP-dependent enhancement of the endothelial barrier, leading to hypo-permeability. We provide the first evidence that endothelial cells and mural cells cooperate to modulate vascular permeability.
PMCID: PMC4294111  PMID: 24923772
3.  PDGF and TGF-β promote tenascin-C expression in subepithelial myofibroblasts and contribute to intestinal mucosal protection in mice 
British Journal of Pharmacology  2013;171(2):375-388.
Background and Purpose: Tenascin-C (TnC) is a multi-domain extracellular matrix glycoprotein that is expressed at a high level during embryogenesis but is almost absent during normal postnatal life. This multi-domain complex molecule is reported to associate with both pro-inflammatory and anti-inflammatory signalling cascades. In this study, we examined how TnC modulated intestinal inflammation.
Experimental Approach: TnC pathophysiology was evaluated in cultures of rat intestinal subepithelial myofibroblasts (ISEMF) and intestinal epithelial cells. Wild-type and TnC(−/−) mice were treated with dextran sodium sulfate (DSS) to induce colitis.
Key Results: DSS-induced colitis in mice markedly increased TnC in the damaged mucosal areas and up-regulated mRNA for TnC, pro-inflammatory cytokines and growth factors (PDGF-B and TGF-β1). In addition, 2,4,6-trinitrobenzene sulfonic acid-induced colitis and SAMP1/Yit mice, a model of spontaneous Crohn's disease, also exhibited increased mucosal TnC in colon and ilea respectively. PDGF receptor-α (PDGFRα) positive ISEMF were the primary TnC-producing cells in colon tissues. Accordingly, ISEMF collected from the rat colon constitutively expressed both TnC and PDGFRα. PDGF-BB and TGF-β1 up-regulated both TnC mRNA and protein levels in ISEMF. Knock-down of TnC gene increased susceptibility to DSS-induced colitis, compared with TnC(+/+) littermates. TnC(−/−) mice showed marked abrasion of intestinal mucosal barrier and increased inflammatory scores. Moreover, TnC accelerated both trans-well migration and wound healing in epithelial cells.
Conclusions and Implications: The pharmacological profiles of PDGF-BB and TGF-β in colitis tissues and ISEMF suggest that increased TnC production during inflammation contributed to epithelial cell migration, remodelling and protection of intestinal barriers.
PMCID: PMC3904258  PMID: 24116743
ISEMF; epithelial cell; tenascin-C; PDGF; TGF-β; intestinal inflammation
4.  Oral administration of an HSP90 inhibitor, 17-DMAG, intervenes tumor-cell infiltration into multiple organs and improves survival period for ATL model mice 
Blood Cancer Journal  2013;3(8):e132-.
In the peripheral blood leukocytes (PBLs) from the carriers of the human T-lymphotropic virus type-1 (HTLV-1) or the patients with adult T-cell leukemia (ATL), nuclear factor kappaB (NF-κB)-mediated antiapoptotic signals are constitutively activated primarily by the HTLV-1-encoded oncoprotein Tax. Tax interacts with the I κB kinase regulatory subunit NEMO (NF-κB essential modulator) to activate NF-κB, and this interaction is maintained in part by a molecular chaperone, heat-shock protein 90 (HSP90), and its co-chaperone cell division cycle 37 (CDC37). The antibiotic geldanamycin (GA) inhibits HSP90's ATP binding for its proper interaction with client proteins. Administration of a novel water-soluble and less toxic GA derivative, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), to Tax-expressing ATL-transformed cell lines, C8166 and MT4, induced significant degradation of Tax. 17-DMAG also facilitated growth arrest and cellular apoptosis to C8166 and MT4 and other ATL cell lines, although this treatment has no apparent effects on normal PBLs. 17-DMAG also downregulated Tax-mediated intracellular signals including the activation of NF-κB, activator protein 1 or HTLV-1 long terminal repeat in Tax-transfected HEK293 cells. Oral administration of 17-DMAG to ATL model mice xenografted with lymphomatous transgenic Lck-Tax (Lck proximal promoter-driven Tax transgene) cells or HTLV-1-producing tumor cells dramatically attenuated aggressive infiltration into multiple organs, inhibited de novo viral production and improved survival period. These observations identified 17-DMAG as a promising candidate for the prevention of ATL progression.
PMCID: PMC3763384  PMID: 23955587
17-DMAG; molecular chaperon; Tax; ATL; apoptosis; transgenic model
5.  Dynamic alteration of regional cerebral blood flow during carotid compression and proof of reversibility 
Acta Radiologica Short Reports  2012;1(2):arsr.2012.110015.
It is difficult to non-invasively visualize changes in regional cerebral blood flow caused by manual compression of the carotid artery.
To visualize dynamic changes in regional cerebral blood flow during and after manual compression of the carotid artery.
Material and Methods
Two healthy volunteers were recruited. Anatomic features and flow directions in the circle of Willis were evaluated with time-of-flight magnetic resonance angiography (MRA) and two-dimensional phase-contrast (2DPC) MRA, respectively. Regional cerebral blood flow was visualized with territorial arterial spin-labeling magnetic resonance imaging (TASL-MRI). TASL-MRI and 2DPC-MRA were performed in three states: at rest, during manual compression of the right carotid artery, and after decompression. In one volunteer, time-space labeling inversion pulse (Time-SLIP) MRA was performed to confirm collateral flow.
During manual carotid compression, in one volunteer, the right thalamus changed to be fed only by the vertebrobasilar system, and the right basal ganglia changed to be fed by the left internal carotid artery. In the other volunteer, the right basal ganglia changed to be fed by the vertebrobasilar system. 2DPC-MRA showed that the flow direction changed in the right A1 segment of the anterior cerebral artery and the right posterior communicating artery. Perfusion patterns and flow directions recovered after decompression. Time-SLIP MRA showed pial vessels and dural collateral circulation when the right carotid artery was manually compressed.
Use of TASL-MRI and 2DPC-MRA was successful for non-invasive visualization of the dynamic changes in regional cerebral blood flow during and after manual carotid compression.
PMCID: PMC3738342  PMID: 23986833
Territorial arterial spin-labeling magnetic resonance imaging (TASL-MRI); two-dimensional phase-contrast MRA (2DPC-MRA); regional cerebral blood flow; manual carotid artery compression
6.  First observation of two hyperfine transitions in antiprotonic 3He 
Physics Letters. [Part B]  2011;700(1):1-6.
We report on the first experimental results for microwave spectroscopy of the hyperfine structure of p¯3He+. Due to the helium nuclear spin, p¯3He+ has a more complex hyperfine structure than p¯4He+, which has already been studied before. Thus a comparison between theoretical calculations and the experimental results will provide a more stringent test of the three-body quantum electrodynamics (QED) theory. Two out of four super-super-hyperfine (SSHF) transition lines of the (n,L)=(36,34) state were observed. The measured frequencies of the individual transitions are 11.12559(14) GHz and 11.15839(18) GHz, less than 1 MHz higher than the current theoretical values, but still within their estimated errors. Although the experimental uncertainty for the difference of these frequencies is still very large as compared to that of theory, its measured value agrees with theoretical calculations. This difference is crucial to be determined because it is proportional to the magnetic moment of the antiproton.
PMCID: PMC3149286  PMID: 21822351
Antiprotonic helium; Microwave spectroscopy; Hyperfine structure; Three-body QED
7.  Bug22p, a Conserved Centrosomal/Ciliary Protein Also Present in Higher Plants, Is Required for an Effective Ciliary Stroke in Paramecium ▿ † 
Eukaryotic Cell  2010;9(4):645-655.
Centrioles, cilia, and flagella are ancestral conserved organelles of eukaryotic cells. Among the proteins identified in the proteomics of ciliary proteins in Paramecium, we focus here on a protein, Bug22p, previously detected by cilia and basal-body high-throughput studies but never analyzed per se. Remarkably, this protein is also present in plants, which lack centrioles and cilia. Bug22p sequence alignments revealed consensus positions that distinguish species with centrioles/cilia from plants. In Paramecium, antibody and green fluorescent protein (GFP) fusion labeling localized Bug22p in basal bodies and cilia, and electron microscopy immunolabeling refined the localization to the terminal plate of the basal bodies, the transition zone, and spots along the axoneme, preferentially between the membrane and the microtubules. RNA interference (RNAi) depletion of Bug22p provoked a strong decrease in swimming speed, followed by cell death after a few days. High-speed video microscopy and morphological analysis of Bug22p-depleted cells showed that the protein plays an important role in the efficiency of ciliary movement by participating in the stroke shape and rigidity of cilia. The defects in cell swimming and growth provoked by RNAi can be complemented by expression of human Bug22p. This is the first reported case of complementation by a human gene in a ciliate.
PMCID: PMC2863418  PMID: 20118210
8.  Prostaglandin E2 induces contraction of liver myofibroblasts by activating EP3 and FP prostanoid receptors 
British Journal of Pharmacology  2009;156(5):835-845.
Background and purpose:
Increased portal pressure in liver injury results from hypercontraction of perivascular non-parenchymal cells including liver myofibroblasts (MFs). Prostaglandin E2 (PGE2) is the major eicosanoid which is released around the venous system during liver injury, but little is known about their contractile effect on MFs.
Experimental approach:
Contraction of primary rat liver MFs was measured by a collagen gel contraction assay. Expression of E prostanoid (EP) receptor subtypes was assessed by reverse transcription-polymerase chain reaction. Fura-2 fluorescence was used to determine intracellular Ca2+ concentration ([Ca2+]i). Phosphorylation of protein kinase C (PKC) was detected by Western blot analysis.
Key results:
Liver MFs expressed mRNAs for all four EP receptors. PGE2 induced contraction in a dose- and time-dependent manner, and slightly increased [Ca2+]i only at high concentrations (10 µmol·L−1). An agonist selective for EP3 receptors, ONO-AE-248, dose-dependently induced MF contraction but did not increase [Ca2+]i. Pretreatment with rottlerin (a specific novel PKC inhibitor) and Ro 31-8425 (a general PKC inhibitor) significantly reduced 1 µmol·L−1 PGE2- or ONO-AE-248-induced contractions. Furthermore, 1 µmol·L−1 PGE2 stimulated phosphorylation of PKC isoforms PKCδ and PKCε. The F prostanoid (FP) receptor antagonist AL8810 abolished the [Ca2+]i elevation and the rapid contraction induced by 10 µmol·L−1 PGE2.
Conclusions and implications:
Lower concentrations up to 1 µmol·L−1 of PGE2 induce liver MF contraction via a [Ca2+]i-independent PKC-mediated pathway through the EP3 receptor, while higher concentrations have an additional pathway leading to Ca2+-dependent contraction through activating the FP receptor.
PMCID: PMC2697760  PMID: 19239477
liver myofibroblast; prostaglandin E2; EP3 receptor; FP receptor; protein kinase C
9.  Anti-inflammatory effects of phytosteryl ferulates in colitis induced by dextran sulphate sodium in mice 
British Journal of Pharmacology  2008;154(4):812-824.
Background and purpose:
We have recently reported that phytosteryl ferulates isolated from rice bran inhibit nuclear factor-κB (NF-κB) activity in macrophages. In the present study, we investigated the effect of γ-oryzanol (γ-ORZ), a mixture of phytosteryl ferulates, cycloartenyl ferulate (CAF), one of the components of γ-ORZ, and ferulic acid (FA), a possible metabolite of γ-ORZ in vivo, on a model of colitis in mice.
Experimental approach:
We induced colitis with dextran sulphate sodium (DSS) in mice and monitored disease activity index (DAI), histopathology score, tissue myeloperoxidase (MPO) activity, mRNA expressions of cytokines and COX-2, colon length, antioxidant potency and NF-κB activity in colitis tissue.
Key results:
Both DAI and histopathology score revealed that DSS induced a severe mucosal colitis, with a marked increase in the thickness of the muscle layer, distortion and loss of crypts, depletion of goblet cells and infiltration of macrophages, granulocytes and lymphocytes. MPO activity, pro-inflammatory cytokines and COX-2 levels, NF-κB p65 nuclear translocation and inhibitory protein of nuclear factor-κB-α degradation levels were significantly increased in DSS-induced colitis tissues. γ-ORZ (50 mg kg−1 day−1 p.o.) markedly inhibited these inflammatory reactions and CAF had a similar potency. In vitro assay demonstrated that γ-ORZ and CAF had strong antioxidant effects comparable to those of α-tocopherol.
Conclusions and implications:
Phytosteryl ferulates could be new potential therapeutic and/or preventive agents for gastrointestinal inflammatory diseases. Their anti-inflammatory effect could be mediated by inhibition of NF-κB activity, which was at least partly due to the antioxidant effect of the FA moiety in the structure of phytosteryl ferulates.
PMCID: PMC2439859  PMID: 18536734
phytosteryl ferulates; γ-oryzanol; cycloartenyl ferulate; colitis; pro-inflammatory cytokines; NF-κB; antioxidant
10.  Serial changes in plasma brain natriuretic peptide concentration at the infarct and non-infarct sites in patients with left ventricular remodelling after myocardial infarction 
Heart  2005;91(12):1573-1577.
Objectives: To clarify the role of infarct and non-infarct sites on left ventricular (LV) remodelling after myocardial infarction by measuring brain natriuretic peptide (BNP) from each site.
Methods and results: BNP from the aorta and the anterior interventricular vein (AIV) was measured in 45 patients with first anterior myocardial infarction at one, six, and 18 months. The LV was significantly dilated (> 10 ml/m2 of end diastolic volume from one to 18 months) in 20 patients (remodelling (R) group) but not in 25 others (non-remodelling (NR) group). Patient characteristics and LV functions did not differ significantly at one month but plasma BNP concentration was higher in group R than in group NR (336 (288) v 116 (106) pg/ml, p < 0.01), predicting the degree of LV dilatation. The difference in BNP concentration between the aortic root and AIV (ΔBNP), reflecting BNP secreted from the infarct site, did not differ at one month. In both groups BNP and ΔBNP significantly decreased from one to six months (p < 0.05) and decreased from six months to 18 months, but the change was not significant. BNP and ΔBNP were significantly higher in group R than in group NR after six months, when LV dilatation was not evident in both groups.
Conclusion: Enhanced BNP secretion at one month in the non-infarct and infarct ventricular sites predicts subsequent LV dilatation (that is, remodelling). The slower process of LV remodelling decreased BNP secretion at both sites. Thus, BNP concentration should be useful for monitoring ventricular remodelling after infarction.
PMCID: PMC1769221  PMID: 15774610
brain natriuretic peptide; anterior myocardial infarction; left ventricular dilatation; remodelling; prediction
11.  Different profiles of Ca2+ responses to endothelin-1 and PDGF in liver myofibroblasts during the process of cell differentiation 
British Journal of Pharmacology  2007;151(6):816-827.
Background and purpose:
Hepatic stellate cells play an important role in liver fibrosis but little is known about liver myofibroblasts located around the central vein and in the portal area. In this study, intracellular Ca2+ concentration ([Ca2+]i) was measured to assess the response to endothelin-1 (ET-1), platelet derived growth factor (PDGF) and ATP in rat liver myofibroblasts.
Experimental approach:
Rat liver myofibroblasts were compared in ‘quiescent' (cultured on Matrigel-coated dishes) and ‘activated' (cultured on non-coated plastic dishes) conditions. [Ca2+]i was measured with the fluorescent dye fura-2 and mRNA for ET-1, PDGF and their receptors by RT-PCR.
Key results:
ET-1 increased [Ca2+]i in quiescent cells but not in activated cells, whereas PDGF-BB increased [Ca2+]i in activated cells but not in quiescent cells. However, there was no difference between responses to ATP in quiescent or activated cells. ET-1 (in quiescent cells), PDGF-BB (in activated cells) and ATP (in both cells) all induced transient increases in [Ca2+]i in the absence of extracellular Ca2+ (with EGTA), indicating the involvement of Ca2+ release from intracellular Ca2+ stores. The sustained increase in [Ca2+]i in the presence of external Ca2+ in activated cells (ATP and PDGF) was significantly reduced by nicardipine, a L-type Ca2+ channel blocker, but not in quiescent cells (ATP and ET-1).
Conclusions and implications:
The different pharmacological profiles of [Ca2+]i-response in quiescent and activated myofibroblasts suggest that ET-1 and PDGF contribute differently to myofibroblast activation during the process of liver fibrosis.
PMCID: PMC2014126  PMID: 17533428
liver myofibroblast; endothelin; PDGF; Ca2+ signalling
12.  Variation during the week in the incidence of acute myocardial infarction: increased risk for Japanese women on Saturdays 
Heart  2003;89(4):398-403.
Background: Variations in the incidence of acute myocardial infarction during the week may differ between and within communities, according to lifestyle.
Objective: To identify potential triggering factors for acute myocardial infarction by examining variations in incidence in the days of the week within the Osaka area of Japan.
Patients: Of 2511 consecutive patients in this region who were admitted to hospital for acute myocardial infarction between April 1998 and March 2001 and consented to take part, 2400 who had a definitely identified time of onset were enrolled.
Results: For this group as a whole, no significant difference in incidence was noted between days of the week. However, in subgroup analyses women were shown to have significant variation through the week, peaking on Saturday with a 39% increase in relative risk (p = 0.037); working subjects showed a peak on Monday, with a 26% increase in relative risk (p = 0.038). Stratified analyses showed that in working men there was a prominent Monday peak in the onset of infarction, with a 30% increase in relative risk (p = 0.022), while in working women, there was no significant variation through the week.
Conclusions: Earlier findings of a Monday peak linked to increased physical and mental occupational stress are confirmed. There is also an increase in uncertain risk factors on Saturdays for Japanese women, possibly involving a stressful weekend burden for women. Confirmation of this finding in other communities may help identify triggers of acute myocardial infarction and be useful in prevention.
PMCID: PMC1769260  PMID: 12639867
acute myocardial infarction; weekly variation; occupational stress; Japanese women
13.  Increased smooth muscle contractility of intestine in the genetic null of the endothelin ETB receptor: a rat model for long segment Hirschsprung's disease 
Gut  2002;50(3):355-360.
Background and aims: The endothelin ETB receptor null rat (ETB(−/−)R) has an intestinal segment without ganglia, and this rat is characterised by intestinal obstruction similar to that observed in human Hirschsprung's disease. In the present study, we have examined the myogenic mechanism responsible for obstruction in the ETB(−/−)R.
Results: The ETB(−/−)R had an enlarged belly and the average lifespan was 18.1 days. The bowel from the rectum to the lower part of the small ileum was constricted whereas the upper region was dilated with faecal stasis and thus presented as megaileum. The constricted muscle segments without ganglia had a greater increase in absolute force when stimulated by carbachol, high K+, and endothelin-1 compared with that of normal siblings. In contrast, in the dilated part with ganglia, the absolute contractile force due to these stimulants in the ETB(−/−)R was not different from that in the ETB(+/+)R. Such a functional hypertrophy of the musculature was observed in parts of the colon, caecum, and distal ileum without ganglia but not in the part of the proximal ileum and jejunum with ganglia. Morphological study demonstrated that the thickness of the circular and longitudinal muscle layers was greater in the constricted part of the intestine in the ETB(−/−)R, and these changes were associated with an increase in the number of smooth muscle cells.
Conclusions: Our findings suggest that both increased contractility of smooth muscle and increased thickness of the intestinal muscular wall may contribute to the intestinal obstruction in the ETB(−/−)R.
PMCID: PMC1773131  PMID: 11839714
endothelin receptor null rat; Hirschsprung's disease; megaileum; smooth muscle
14.  Prognostic significance of cardiac 123I metaiodobenzylguanidine imaging for mortality and morbidity in patients with chronic heart failure: a prospective study 
Heart  2001;86(6):656-660.
OBJECTIVE—To determine whether cardiac iodine-123 metaiodobenzylguanidine (123I MIBG) imaging is useful in predicting the prognosis of patients with chronic heart failure.
DESIGN—Cardiac 123I MIBG imaging was done on entry to the study. The cardiac MIBG washout rate was calculated from anterior chest view images obtained 20 and 200 minutes after injection of the isotope. Study patients were divided into two groups with washout rates above and below 27% (the mean value + 2 SD obtained in 20 normal subjects), and were then followed up.
SETTING—Tertiary referral centre.
PATIENTS—79 patients with chronic heart failure in whom the left ventricular ejection fraction was less than 40%.
RESULTS—There were 37 patients in group 1 (washout rate of ⩾ 27%) and 42 in group 2 (< 27%). During a follow up period of between 1 and 52 months, eight patients died suddenly and five died of worsening heart failure in group 1, while none died in group 2; 13 patients in group 1 and four in group 2 were admitted to hospital for progressive heart failure. Kaplan-Meier analysis showed that group 1 had a significantly higher mortality and morbidity (p = 0.001 and p < 0.001, respectively) than group 2.
CONCLUSIONS—Cardiac 123I MIBG washout rate seems to be a good predictor of prognosis in patients with chronic heart failure.

Keywords: chronic heart failure; sudden death; cardiac adrenergic nerve activity
PMCID: PMC1730035  PMID: 11711461
15.  Sustained left ventricular diastolic dysfunction after exercise in patients with dilated cardiomyopathy 
Heart  1998;80(3):263-269.
Objective—To investigate the recovery process of exercise induced diastolic dysfunction in heart failure, using Doppler echocardiographic techniques.
Design and patients—Transmitral flow velocity profiles and standard non-invasive haemodynamic indices were obtained serially over seven days after symptom limited bicycle exercise tests in 18 patients with dilated cardiomyopathy and eight normal subjects. In three patients with cardiomyopathy we also measured the pulmonary capillary wedge pressure for 24 hours after exercise.
Results—The intensity of exercise, as assessed by respiratory gas analysis, was lower in patients with dilated cardiomyopathy than in normal subjects. Despite the higher exercise level, all haemodynamic variables returned to baseline within one hour after exercise in normal subjects. In contrast, patients with dilated cardiomyopathy showed a sustained decrease in the peak early diastolic filling velocity and a sustained increase in the deceleration time of early filling for 24 hours or more after exercise. Because other haemodynamic variables recovered within one hour after exercise even in patients with dilated cardiomyopathy, the postexercise changes in ventricular filling were not explained by changes in loading conditions.
Conclusions—Exercise induced diastolic left ventricular dysfunction of the failing heart persists for 24 hours or more after exercise. The efficacy of exercise training on a daily basis in dilated cardiomyopathy requires further evaluation.

 Keywords: exercise;  chronic heart failure;  mitral flow velocity;  diastolic stunning
PMCID: PMC1761094  PMID: 9875086
16.  Prediction of the effectiveness of long term β blocker treatment for dilated cardiomyopathy by signal averaged electrocardiography 
Heart  1998;79(3):256-261.
Objective—To determine whether the effectiveness of long term β blocker treatment for idiopathic dilated cardiomyopathy can be predicted by signal averaged electrocardiography (ECG).
Patients—31 patients with dilated cardiomyopathy and without bundle branch block were included in a retrospective study and 16 in a prospective study.
Methods—A signal averaged ECG was recorded before β blocker treatment, and three variables were measured from the vector magnitude: QRS duration, root mean square voltage for the last 40 ms (RMS40), and duration of the terminal low amplitude signals (< 40 µV) (LAS40). In the retrospective study, these variables were compared among good responders (showing ⩾ 0.10 increase in ejection fraction 12 months after start of β blocker treatment) and poor responders without such improvement. The validity of the signal averaged ECG criteria for prediction of the response to β blocker treatment was examined in the prospective study.
Results—In the retrospective study, good responders (n = 16) had a shorter QRS duration (mean (SD): 122.9 (11) v 138 (14.4) ms, p < 0.005) and LAS40 (33.1 (8.9) v 42.5 (7.8) ms, p < 0.005), and a higher RMS40 (31.6 (16.3) v 19.0 (10.3) µV, p < 0.02) than poor responders (n = 15). Signal averaged ECG criteria for good response were defined as two or more of the following: QRS duration < 130 ms, RMS40 > 20 µV, LAS40 < 40 ms (sensitivity 81%, specificity 73%). In the prospective study, six of seven patients who met these criteria showed a good response to the β blocker treatment, while eight of nine who did not showed a poor response (χ2 = 6.1, p < 0.02). The signal averaged ECG criteria gave a sensitivity of 86% and a specificity of 89% for predicting the effectiveness of β blocker treatment.
Conclusions—A signal averaged ECG might be useful in predicting the effectiveness of β blocker treatment for dilated cardiomyopathy.

 Keywords: signal averaged ECG;  β blockers;  dilated cardiomyopathy
PMCID: PMC1728625  PMID: 9602659
17.  Endogenous adenosine inhibits P-selectin-dependent formation of coronary thromboemboli during hypoperfusion in dogs. 
Journal of Clinical Investigation  1998;101(8):1643-1653.
The activation of platelets and the formation of neutrophil- platelet conjugates may lead to the development of thromboemboli. We studied whether blockade of adenosine receptors during coronary hypoperfusion may cause thromboemboli via P-selectin-dependent mechanisms in 30 open-chest dogs. When coronary blood flow was reduced to 20% of the control, it was stable at low levels with increases in adenosine levels. When 8-p-sulfophenyltheophylline, an adenosine receptor antagonist, was infused during coronary hypoperfusion, coronary blood flow decreased gradually and approached almost zero 20 min after its administration. Histological examination revealed thromboemboli in the small coronary vessels. During hypoperfusion in the presence of 8-p-sulfophenyltheophylline, the mAb against P-selectin attenuated both the reduction in coronary blood flow and the formation of thromboemboli, and improved contractile and metabolic dysfunction of the myocardium. Flow cytometric analysis indicated that the expression of P-selectin on platelet and neutrophil-platelet adhesion were increased during coronary hypoperfusion, and that both were further augmented by 8-p-sulfophenyltheophylline. Immunohistochemical examination showed no staining of P-selectin in the ischemic myocardium. Adenosine inhibited the thrombin-induced expression of P-selectin on platelet and neutrophil- platelet adhesion via adenosine A2 receptors. Adenosine appears to inhibit the formation of thromboemboli during coronary hypoperfusion by suppressing the expression of P-selectin on platelets and neutrophil-platelet adhesion.
PMCID: PMC508745  PMID: 9541494
18.  Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells. 
Journal of Clinical Investigation  1997;99(12):2961-2970.
It has been reported that PTH exerts bone-forming effects in vivo when administered intermittently. In the present study, the anabolic effects of PTH(1-34) on osteoblast differentiation were examined in vitro. Osteoblastic cells isolated from newborn rat calvaria were cyclically treated with PTH(1-34) for the first few hours of each 48-h incubation cycle. When osteoblastic cells were intermittently exposed to PTH only for the first hour of each 48-h incubation cycle and cultured for the remainder of the cycle without the hormone, osteoblast differentiation was inhibited by suppressing alkaline phosphatase activity, bone nodule formation, and mRNA expression of alkaline phosphatase, osteocalcin, and PTH/PTHrP receptor. Experiments using inhibitors and stimulators of cAMP/protein kinase A (PKA) and Ca2+/PKC demonstrated that cAMP/PKA was the major signal transduction system in the inhibitory action of PTH. In contrast, the intermittent exposure to PTH for the first 6 h of each 48-h cycle stimulated osteoblast differentiation. Both cAMP/ PKA and Ca2+/PKC systems appeared to be involved cooperatively in this anabolic effect. Continuous exposure to PTH during the 48-h incubation cycle strongly inhibited osteoblast differentiation. Although both cAMP/PKA and Ca2+/PKC were involved in the effect of continuous exposure to PTH, they appeared to act independently. A neutralizing antibody against IGF-I blocked the stimulatory effect on alkaline phosphatase activity and the expression of osteocalcin mRNA induced by the 6-h intermittent exposure. The inhibitory effect induced by the 1-h intermittent exposure was not affected by anti-IGF-I antibody. These results suggest that PTH has diverse effects on osteoblast differentiation depending on the exposure time in vitro mediated through different signal transduction systems. These in vitro findings explain at least in part the in vivo action of PTH that varies with the mode of administration.
PMCID: PMC508148  PMID: 9185520
19.  Dissociation of P2 purinoceptor-mediated increase in intracellular Ca2+ level from myosin light chain phosphorylation and contraction in rat aorta. 
British Journal of Pharmacology  1996;118(3):543-548.
1. The effects of P2 agonists, adenosine-5'-triphosphate (ATP), alpha, beta-methylene-adenosine-5'-triphosphate (alpha, beta-me-ATP) and adenosine 5-O-(3-thiotriphosphate) (ATP gamma S), on the intracellular free Ca2+ level ([Ca2+]i), myosin light chain (MLC) phosphorylation and force of contraction were examined in vascular smooth muscle of rat aorta. 2. ATP (0.1 microM-1 mM), alpha, beta-me-ATP (0.1-100 microM) and ATP gamma S (1-100 microM) induced transient increases followed by sustained increase in [Ca2+]i. The effects of these agonists were concentration-dependent. Compared with the effects of a high concentration of KCl (17.5-72.4 mM), the contractions induced by these P2 purinoceptor agonists were smaller at a given [Ca2+]i. 3. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), ATP gamma S (10 microM) induced large transient increase in [Ca2+]i with only small contraction in Ca(2+)-free solution. In contrast, alpha, beta-me-ATP (10 microM) induced only a very small increase in [Ca2+]i and contraction. 4. ATP (1 mM), alpha, beta-me-ATP (10 microM) and ATP gamma S (10 microM), added during stimulation with 0.1 microM noradrenaline, induced additional and transient increases in [Ca2+]i which were also not associated with contraction. 5. High K+ (72.4 mM) increased MLC phosphorylation with a similar time course to that of the increase in [Ca2+]i (peak phosphorylation was 56% when [Ca2+]i increased to 100%). In contrast, the time course of the increase in MLC phosphorylation due to ATP (1 mM) did not coincide with that of the large increases in [Ca2+]i; MLC phosphorylation increased to only 31% when [Ca2+]i increased to 163%. The MLC phosphorylation due to alpha, beta-me-ATP (10 microM) and ATP gamma S (10 microM), measured at peak [Ca2+]i, were only 19% and 14%, respectively, irrespective of a large increase in [Ca2+]i (138% and 188%, respectively). 6. The absence of a clear relationship between P2-purinoceptor-mediated increase in [Ca2+]i (either by Ca2+ influx or Ca2+ release) and MLC phosphorylation or force generation appears to imply that elevation in [Ca2+]i does not contribute to these responses.
PMCID: PMC1909735  PMID: 8762076
20.  Intrauterine transmission of human T-cell leukemia virus type I in rats. 
Journal of Virology  1995;69(2):1302-1305.
To analyze intrauterine transmission, MT-2 cells, a human T-cell line producing human T-cell leukemia virus type I (HTLV-I), were injected into eight pregnant F344 rats, and cesarean section was performed at day 23 of pregnancy. HTLV-I provirus was detected by PCR in the liver and spleen taken from one of the eight fetuses. Moreover, 71 offspring were delivered by cesarean section from the remaining seven dams and fostered by seven normal rats. HTLV-I provirus was detected in peripheral blood mononuclear cells in 2 of the 71 offspring 4 weeks after cesarean section. These results indicate for the first time the intrauterine transmission of HTLV-I. To confirm the postnatal transmission, MT-2 cells were injected into a dam within 24 h after delivery, and six offspring were fostered by this dam. HTLV-I provirus was detected in peripheral blood mononuclear cells of all six offspring. This animal model may be useful for analysis and prevention of mother-to-child transmission of HTLV-I.
PMCID: PMC188709  PMID: 7815510
21.  Different pathways of calcium sensitization activated by receptor agonists and phorbol esters in vascular smooth muscle. 
British Journal of Pharmacology  1993;110(4):1527-1531.
1. It has been shown that receptor agonists and activators of protein kinase C, phorbol esters, increase Ca2+ sensitivity of contractile elements in vascular smooth muscle. To discover if protein kinase C is involved in the agonist-mediated Ca2+ sensitization, we examined the effects of receptor agonists in the rat isolated aorta in which protein kinase C activity had been diminished by pretreatment with phorbol 12-myristate 13-acetate for 24 h. 2. In the aorta with protein kinase C activity, a high concentration (1 microM) of 12-deoxyphorbol 13-isobutyrate induced contraction and a low concentration (100 nM) potentiated high K(+)-induced contraction. In addition, prostaglandin F2 alpha induced greater contractions than high K+ at a given cytosolic Ca2+ level. The maximally effective concentrations of noradrenaline and endothelin-1 also induced greater contraction than high K+. In the aorta without protein kinase C activity, the contraction induced by 12-deoxyphorbol 13-isobutyrate and its potentiation of the high K(+)-induced contraction were abolished. However, prostaglandin F2 alpha, noradrenaline and endothelin-1 still induced a greater contraction than high K+. 3. In the aorta without protein kinase C activity, noradrenaline, endothelin-1 and prostaglandin F 2 alpha, but not 12-deoxyphorbol 13-isobutyrate, induced contractions in the presence of the Ca2+ channel blocker, verapamil, or in the absence of external Ca2+, by increasing Ca2+ sensitivity. 4. In the permeabilized preparations, inhibition of protein kinase C activity abolished the effect of potentiation of the Ca(2+)-induced contraction by 12-deoxyphorbol 13-isobutyrate although the potentiation of the contraction by prostaglandin F2 alpha did not change.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC2175865  PMID: 8306097
22.  Evidence for deactivation of both ectosolic and cytosolic 5'-nucleotidase by adenosine A1 receptor activation in the rat cardiomyocytes. 
Journal of Clinical Investigation  1994;94(6):2451-2456.
Adenosine, an important regulator of many cardiac functions, is produced by ectosolic and cytosolic 5'-nucleotidase. The activity of these enzymes is influenced by several ischemia-sensitive metabolic factors, e.g., ATP, ADP, H+, and inorganic phosphate. However, there is no clear evidence that adenosine itself affects 5'-nucleotidase activity. This study tested whether adenosine decreases the activity of ectosolic and cytosolic 5'-nucleotidase. Cardiomyocytes were isolated from adult male Wistar rats and suspended in the modified Hepes-Tyrode buffer solution. After stabilization, isolated cardiomyocytes were incubated with and without adenosine (10(-9) - 10(-4) M). Ectosolic and cytosolic 5'-nucleotidase activity was decreased by exogenous adenosine (ectosolic 5'-nucleotidase activity, 20.6 +/- 2.3 vs. 8.6 +/- 1.6 mumol/min per 10(6) cells [P < 0.05]; cytosolic 5'-nucleotidase activity, 2.47 +/- 0.58 vs. 1.61 +/- 0.54 mumol/min per 10(6) cells [P < 0.05] at 10(-6) M adenosine) after 30 min. The decrease in ectosolic and cytosolic 5'-nucleotidase activity was inhibited by 8-phenyltheophylline and pertussis toxin, and was mimicked by N6-cyclohexyladenosine, an adenosine A1 receptor agonist. Neither CGS21680C, and A2 receptor agonist, nor cycloheximide deactivated ectosolic and cytosolic 5'-nucleotidase. Thus, we conclude that activation of adenosine A1 receptors is coupled to Gi proteins and attenuates ectosolic and cytosolic 5'-nucleotidase activity in rat cardiomyocytes.
PMCID: PMC330077  PMID: 7989602
23.  Induction of manganese superoxide dismutase in rat cardiac myocytes increases tolerance to hypoxia 24 hours after preconditioning. 
Journal of Clinical Investigation  1994;94(6):2193-2199.
Manganese superoxide dismutase (Mn-SOD) is induced in ischemic hearts 24 h after ischemic preconditioning, when tolerance to ischemia is acquired. We examined the relationship between Mn-SOD induction and the protective effect of preconditioning using cultured rat cardiac myocytes. Exposure of cardiac myocytes to brief hypoxia (1 h) decreased creatine kinase release induced by sustained hypoxia (3 h) that follows when the sustained hypoxia was applied 24 h after hypoxic preconditioning (57% of that in cells without preconditioning). The activity and content of Mn-SOD in cardiac myocytes were increased 24 h after hypoxic preconditioning (activity, 170%; content, 139% compared with cells without preconditioning) coincidentally with the acquisition of tolerance to hypoxia. Mn-SOD mRNA was also increased 20-40 min after preconditioning. Antisense oligodeoxyribonucleotides corresponding to the initiation site of Mn-SOD translation inhibited the increases in the Mn-SOD content and activity and abolished the expected decrease in creatine kinase release induced by sustained hypoxia after 24 h of hypoxic preconditioning. Sense oligodeoxyribonucleotides did not abolish either Mn-SOD induction or tolerance to hypoxia. These results suggest that the induction of Mn-SOD in myocytes by preconditioning plays a pivotal role in the acquisition of tolerance to ischemia at a later phase (24 h) of ischemic preconditioning.
PMCID: PMC330044  PMID: 7989574
24.  High incidence of HAM/TSP-like symptoms in WKA rats after administration of human T-cell leukemia virus type 1-producing cells. 
Journal of Virology  1994;68(11):7221-7226.
We demonstrate a significantly high incidence of human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy (HAM)-or tropical spastic paraparesis (TSP)-like symptoms in WKA rats after injection with HTLV-1-producing MT-2 cells, while no symptoms were observed in F344 rats injected with MT-2 cells or in control WKA rats. Five of the eight (63%) WKA rats injected with MT-2 cells showed HAM/TSP-like paraparesis at 105 weeks of age, but none of seven MT-2-injected F344 rats or eight control WKA rats showed symptoms. This high incidence of HAM/TSP-like symptoms in WKA rats was statistically significant (P < 0.05). Six of the eight (75%) WKA rats injected with MT-2 cells showed HAM/TSP-like paraparesis at 108 weeks of age. HAM/TSP-like symptoms were also observed in one of the two WKA rats injected with HTLV-1-producing Ra-1 cells at 128 weeks of age. HTLV-1 provirus was detected in peripheral blood mononuclear cells in both WKA and F344 rats. The provirus was detected in the spinal cords of the HAM/TSP-like WKA rats that had severe neuropathological changes. WKA and F344 rats showed no significant difference in antibody response against HTLV-1 Gag antigen. However, the antibody response against the C-terminal half of gp46 HTLV-1 envelope protein was lower in WKA rats than in F344 rats. Pathological analysis of the HAM/TSP-like rats showed degeneration of the white matter of the spinal cord and peripheral nerves. These findings suggest that both the genetic background of the host and HTLV-1 infection are important in neuropathogenesis of HAM/TSP-like paraparesis in rats.
PMCID: PMC237161  PMID: 7933104
25.  Induction of endothelium-dependent relaxation in the rat aorta by IRL 1620, a novel and selective agonist at the endothelin ETB receptor. 
British Journal of Pharmacology  1993;109(2):486-490.
1. The effects of a novel and selective agonist at the endothelin ETB receptor, IRL 1620 (Suc-[Glu9, Ala11,15] endothelin-1 (8-21)), were examined in the isolated aorta of the rat. 2. IRL 1620 (1-300 nM) changed neither the resting tone nor the cytosolic Ca2+ level ([Ca2+]i) of the aorta without endothelium. In the presence of endothelium, however, IRL 1620 increased endothelial [Ca2+]i with little effect on the muscle tone. In the absence of external Ca2+, IRL 1620 still induced a transient increase in endothelial [Ca2+]i. 3. Noradrenaline (100 nM) increased both muscle [Ca2+]i and tension. IRL 1620 (1-300 nM) relaxed the muscle with an increase in endothelial [Ca2+]i only in the presence of endothelium. An inhibitor of nitric oxide synthase, 100 microM NG-monomethyl-L-arginine, inhibited the relaxant effect of IRL 1620 but not the increase in endothelial [Ca2+]i. 4. In resting and noradrenaline-stimulated aorta, the effects of IRL 1620 were inhibited by a selective antagonist of the ETB receptor, IRL 1038 (0.3-3 microM), although a selective antagonist of the ETA receptor, BQ-123 (3 microM), was ineffective. Verapamil (10 microM) did not alter the effects of IRL 1620. 5. A muscarinic receptor agonist, carbachol (1 microM), also induced endothelium-dependent relaxation with an increase in endothelial [Ca2+]i. However, the effects of carbachol were not inhibited by the ETB antagonist, IRL 1038 (3 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC2175706  PMID: 8358547

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