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Takaharu Ishibashi

Publications and source records attributed to Takaharu Ishibashi.

16 recordsLinked to original sources
PubMed2006

G1 cell cycle arrest by amlodipine, a dihydropyridine Ca2+ channel blocker, in human epidermoid carcinoma A431 cells.

We demonstrated previously that amlodipine, a dihydropyridine Ca(2+) channel blocker, exhibits antitumor effects on human epidermoid carcinoma A431 cells both in vitro and in vivo, in part through inhibition of capacitative Ca(2+) entry. In this study, we examined the effects of amlodipine on cell cycle distribution and cell cycle regulatory molecules in A431 cells, since a rise in intracellular Ca(2+) is required at several points during cell cycle progression. Flow cytometric analysis revealed that treatment with amlodipine (20-30muM, for 24h) induced G1 phase cell accumulation. The amlodipine-induced G1 arrest was associated with a decrease in phosphorylation of retinoblastoma protein (pRB), a regulator of G1 to S phase transition, reduction of protein levels of cyclin D1 and cyclin dependent kinase 4 (CDK4), G1 specific cell cycle proteins, and increased expression of p21(Waf1/Cip1), an inhibitory protein of CDK/cyclin complexes. In vitro kinase assay revealed that amlodipine significantly decreased CDK2-, CDK4-, and their partners cyclin E- and cyclin D1-associated kinase activities. The amlodipine-induced reductions in cyclin D1 protein expression and in CDK2 kinase activity were reproduced by a dihydropyridine derivative, nicardipine, having an inhibitory effect on A431 cell growth, but not by nifedipine, lacking the antiproliferative activity. Our results demonstrate that amlodipine caused G1 cell cycle arrest and growth inhibition in A431 cells through induction of p21(Waf1/Cip1) expression, inhibition of CDK/cyclin-associated kinase activities, and reduced phosphorylation of pRB.

Amlodipine↗
PubMed2006

N(G)-nitro-L-arginine methyl ester potentiates anaphylactic venoconstriction in rat perfused livers.

1. The effects of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on anaphylaxis-induced venoconstriction were examined in rat isolated livers perfused with blood-free solutions in order to clarify the role of NO in anaphylactic venoconstriction. 2. Rats were sensitized with ovalbumin (1 mg) and, 2 weeks later, livers were excised and perfused portally in a recirculating manner at a constant flow with Krebs'-Henseleit solution. The antigen (ovalbumin; 0.1 mg) was injected into the reservoir 10 min after pretreatment with L-NAME (100 micromol/L) or D-NAME (100 micromol/L) and changes in portal vein pressure (Ppv), hepatic vein pressure (Phv) and perfusate flow were monitored. In addition, concentrations of the stable metabolites of NO ( and ) were determined in the perfusate using an HPLC-Griess system. 3. The antigen caused hepatic venoconstriction, as evidenced by an increase in Ppv from a mean (SEM) baseline value of 7.7 +/- 0.1 cmH2O to a peak of 21.4 +/- 1.1 cmH2O at 3 min in D-NAME-pretreated livers. Pretreatment with L-NAME augmented anaphylactic venoconstriction, as reflected by a higher Ppv (27.4 +/- 0.8 cmH2O) after antigen than observed following D-NAME pretreatment. The addition of L-arginine, a precursor for the synthesis of NO, reversed the augmentation of anaphylactic venoconstricion by L-NAME. This suggests that hepatic anaphylaxis increased the production of NO, which consequently attenuated anaphylactic venoconstriction. However, perfusate NOx levels did not increase significantly after antigen in livers pretreated with either L-NAME or D-NAME. 4. In conclusion, L-NAME potentiates rat anaphylactic hepatic venoconstriction, suggesting that NO contributes to the attenuation of the venoconstriction. However, this functional evidence was not accompanied by corresponding changes in perfusate NOx concentrations.

Anaphylaxis↗
PubMed2005

Capacitative Ca2+ entries and mRNA expression for TRPC1 and TRPC5 channels in human epidermoid carcinoma A431 cells.

In human epidermoid carcinoma A431 cells, capacitative Ca2+ entries in response to intracellular Ca2+ store depletion with thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, and uridine 5'-triphosphate, a phospholipase C-linked agonist, were inhibited by trivalent cations such as Gd3+ and La3+, and by the store-operated Ca2+ channel inhibitor, 2-aminoethoxydiphenyl borate. Of the seven types of canonical transient receptor potential (TRPC) channels as molecular candidates for store-operated Ca2+ channels, mRNAs for TRPC1 and TRPC5 were detected in the cells with the reverse transcription-polymerase chain reaction. Western blotting confirmed the protein expressions of TRPC1 and TRPC5 in A431 cells. The present results suggest that TRPC1 and/or TRPC5 channels serve as store-operated Ca2+ channels in A431 cells, and may function as regulators for intracellular Ca2+ signaling.

Base Sequence↗
PubMed2004

Antitumor effects of amlodipine, a Ca2+ channel blocker, on human epidermoid carcinoma A431 cells in vitro and in vivo.

Amlodipine, a dihydropyridine Ca(2+) channel blocker, is reported to inhibit proliferation of human epidermoid carcinoma A431 cells, and specifically attenuates Ca(2+) responses evoked by thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPases. In this study, we further examined the possible mechanism of the antiproliferative action of amlodipine and its antitumor effect on A431 xenografts in nude mice. Amlodipine reduced BrdU incorporation into nucleic acids in serum-starved A431 cells, and the reduction was diminished by uridine 5'-triphosphate (UTP), a phospholipase C (PLC)-linked agonist. Fluorometric measurement of intracellular free Ca(2+) concentration revealed that amlodipine blunted the UTP-induced Ca(2+) release from the internal Ca(2+) stores and consequently Ca(2+) influx through Ca(2+)-permeable channels on the plasma membrane. Although amlodipine alone caused Ca(2+) release from thapsigargin-sensitive Ca(2+) stores, such an effect was not reproduced by other dihydropyridine Ca(2+) channel blockers, including nicardipine and nimodipine, despite their antiproliferative effects in the cells. Daily intraperitoneal administration of amlodipine (10 mg/kg) for 20 days into mice bearing A431 xenografts retarded tumor growth and prolonged the survival of mice. Our results suggest a potential antitumor action for amlodipine in vitro and in vivo, which may be in part mediated by inhibiting Ca(2+) influx evoked by the passive depletion of internal Ca(2+) stores and by PLC-linked agonist stimulation.

Amlodipine↗
PubMed2004

Malnutrition impairs alveolar fluid clearance in rat lungs.

Inadequate nutrition complicates the clinical course of critically ill patients, and many of these patients develop pulmonary edema. However, little is known about the effect of malnutrition on the mechanisms that resolve alveolar edema. Therefore, we studied the mechanisms responsible for the decrease in alveolar fluid clearance in rats exposed to malnutrition. Rats were allowed access to water, but not to food, for 120 h. Then, the left and right lungs were isolated for the measurement of lung water volume and alveolar fluid clearance, respectively. The rate of alveolar fluid clearance was measured by the progressive increase in the concentration of Evans blue dye that was instilled into the distal air spaces with an isosmolar 5% albumin solution over 1 h. Malnutrition decreased alveolar fluid clearance by 38% compared with controls. Amiloride (10(-3) M) abolished alveolar fluid clearance in malnourished rats. Either refeeding for 120 h following nutritional deprivation for 120 h or an oral supply of sodium glutamate during nutritional deprivation for 120 h restored alveolar fluid clearance to 91 and 86% of normal, respectively. Dibutyryl-cGMP, a cyclic nucleotide-gated cation channel agonist, increased alveolar fluid clearance in malnourished rats supplied with sodium glutamate. Terbutaline, a beta(2)-adrenergic agonist, increased alveolar fluid clearance in rats under all conditions (control, malnutrition, refeeding, and glutamate-treated). These results indicate that malnutrition impairs primarily amiloride-insensitive and dibutyryl-cGMP-sensitive alveolar fluid clearance, but this effect is partially reversible by refeeding, treatment with sodium glutamate, or beta-adrenergic agonist therapy.

Adenosine Triphosphate↗
PubMed2004

Implication of steady state concentrations of nitrite and nitrate metabolites of nitric oxide in plasma and whole blood in healthy human subjects.

1. The aim of the present study was to determine whether the steady state NOx concentration reflects NOx formation in vivo. 2. A NO3- load study was performed after achieving NOx steady state. Chronological changes in NOx concentrations in plasma and whole blood samples from nine healthy subjects were determined by the HPLC-Griess system and NOx concentrations in erythrocytes were estimated as a possible NOx compartment influential in regulating plasma NOx concentrations. 3. Analysis was performed using the first-order one-compartment open model and the NOx formation rate was subsequently calculated. 4. The mean (+/-SEM) steady state NOx concentration of plasma (15.5 +/- 1.6 micromol/L), whole blood (12.8 +/- 1.2 micromol/L) and erythrocytes (11.9 +/- 0.7 micromol/L) did not correlate with the NOx formation rate in the compartments (0.50 +/- 0.05, 0.61 +/- 0.04 and 0.91 +/- 0.17 micromol/kg per h, respectively), whereas a significant correlation was found between the steady state NOx concentration and NOx elimination rate (Kel) in plasma (r=-0.69; P=0.04) and whole blood (r=-0.79; P=0.01). 5. Although there was no direct correlation between steady state NOx concentrations and serum creatinine levels, the correlation between half-life and serum creatinine levels was significant (plasma: r=0.60, P=0.02; whole blood: r=0.49, P=0.04). 6. Plasma NOx concentrations correlated significantly with erythrocyte NOx concentrations (r=0.92, P <0.01; erythrocyte NOx=0.66 x plasma NOx). 7. The results of the present study indicate that NOx does not accumulate excessively into erythrocytes at steady state and during a NO3- load and that the steady state NOx concentration in whole blood and plasma preferentially implies NOx elimination (mainly depending on renal function) rather than NOx formation.

Adult↗
PubMed2003

Antiproliferative effect of Ca2+ channel blockers on human epidermoid carcinoma A431 cells.

The effects of Ca(2+) channel blockers on the proliferation of human epidermoid carcinoma A431 cells were investigated by microtiter tetrazolium (MTT) proliferation assay and bromodeoxyuridine (BrdU) incorporation assay. Dihydropyridine derivatives, such as amlodipine, nicardipine, and nimodipine inhibited A431 cell growth and the incorporation of BrdU into cells with IC(50) values of 20-30 microM, while verapamil, diltiazem and dihydropyridine nifedipine inhibited neither the cell growth nor BrdU incorporation at the same concentration. Though extracellular Ca(2+) is indispensable to the cell growth, an L-type Ca(2+) channel agonist, 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl) phenyl]pyridine-3-carboxylic acid methyl ester (200 nM), did not affect the antiproliferative action of amlodipine. Thapsigargin, an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, inhibited itself the growth of A431 cells and also showed a synergistic effect with the antiproliferative action of amlodipine. In the fluorimetric measurement of intracellular free Ca(2+) concentration in fura-2 or fluo-3 loaded A431 cells, amlodipine blunted the thapsigargin- or cyclopiazonic acid-induced Ca(2+) release from endoplasmic reticulum and the ensuing Ca(2+) influx through Ca(2+)-permeable channels. The effect on the thapsigargin-induced Ca(2+) responses could be reproduced by nicardipine and nimodipine but not by nifedipine or verapamil, lacking antiproliferative potency. These findings suggest that the intracellular Ca(2+) control system responsible for thapsigargin- and cyclopiazonic acid-sensitive endoplasmic reticulum, but not L-type Ca(2+) channels, may be modulated by amlodipine, which results in the inhibition of A431 cell growth.

Calcium↗
PubMed2003

Interference with Saville's method in determination of low-molecular weight S-nitrosothiols by ultrafiltration.

To detect low-molecular weight S-nitrosothiols in human plasma, we used a system combining HPLC for separation and Saville's method for colorimetric detection of S-nitrosothiols. The sensitivity and detection limit was 1-2 nM for both S-nitrosocysteine and S-nitrosoglutathione. When plasma was analyzed after ultrafiltration (with units requiring higher g force [5000 g], irrespective to the material of the membrane) to eliminate high molecular substances, a signal corresponding to S-nitrosoglutahione was recognized. This signal behaved as real S-nitrosoglutathione as it was partially Hg(2+)-sensitive and gradually decayed with time. However, the use of pre-washed units or another ultrafiltration unit that required lower g force (1800 g) or direct application of plasma to the HPLC-Saville's method system did not result in such signal. Based on these observations, it is important to be aware of the interference originating from the ultrafiltration unit and its potential effect on the precise quantification of low molecular weight S-nitrosothiols using Saville's method.

Artifacts↗
PubMed2003

Decrease in plasma NOx concentration by isosorbide dinitrate, an organic nitrate ester.

It has been suggested that isosorbide dinitrate (ISDN)-induced venodilation could be ascribed to preferential accumulation of the agent in venous tissues, resulting in higher concentrations of nitric oxide (NO). Here, the authors investigated whether the venodilating effect of ISDN is associated with a preferential increase in plasma concentrations of NOx (NO2- and NO3-, stable end-products of NO) in venous blood than arterial blood. Plasma NOx was measured by high-performance liquid chromatography-Griess system with a sensitivity of 0.01 microM for NO2- and 0.1 microM for NO3-. Arterial and venous blood samples were obtained after coronary angiography from the aorta and right atrium of patients with or without ischemic heart disease. Nicardipine, a calcium channel blocker, was used as a non-NO-related arteriovasodilator. At 1 mg i.v., it did not cause any changes in NOx concentration in arterial and venous blood irrespective of hemodynamic changes. However, ISDN (3 mg i.v.) increased NO2- and decreased NO3- in both arterial and venous blood, with concomitant venodilation. Further analysis revealed that plasma NO increased in the pulmonary circulation and this increase was preserved after nicardipine and ISDN, and that ISDN, but not nicardipine, increased plasma NO3- in the pulmonary circulation. The authors did not detect higher concentrations of NOx in venous blood relative to their level in arterial blood. Further studies are necessary to clarify the kinetics of NO and NO-related compounds in the whole body.

Aged↗
PubMed2003

Significance of asymmetric dimethylarginine (ADMA) concentrations during coronary circulation in patients with vasospastic angina.

The basal activity of nitric oxide (NO) is reduced in spastic arteries of patients with vasospastic angina (VSA). Elevated concentrations of ADMA are associated with reduced NO production and impaired endothelium-dependent vasodilatation. The aim of this study was to elucidate the role of ADMA and its relationship to NO end-products (NOx; nitrate + nitrite) during coronary circulation in patients with VSA. The plasma ADMA and NOx concentrations during coronary circulation were evaluated in 16 VSA and 16 control patients. Blood samples were obtained from the coronary sinus (V) and the ostium of the left coronary artery (A), and the (V-A) differences of ADMA and NOx were determined. The coronary sinus plasma ADMA concentration in patients with VSA was higher than that in the control. The coronary sinus - arterial (V-A) difference of NOx was negative in the VSA group and approximately zero in the control group (VSA group =-1.4 micromol/L, control group =-0.1 micromol/L, p=0.0005). Furthermore, in the VSA patients, there was a negative correlation between the (V-A) difference of NOx and the basal coronary artery tone at the site of spasm (r=-0.60, p=0.015). A significant negative correlation between the (V-A) differences of NOx and ADMA was observed in patients with VSA (r=-0.52, p<0.05), but not in those of the control. Higher ADMA concentrations might cause the reduced formation of NO that underlies the pathophysiology of coronary vasospasm.

Aged↗
PubMed2003

New methods to evaluate endothelial function: A search for a marker of nitric oxide (NO) in vivo: re-evaluation of NOx in plasma and red blood cells and a trial to detect nitrosothiols.

Although plasma NOx (NO(2)(-) and NO(3)(-)) has been used as an index of nitric oxide (NO) formation in vivo, many unreasonable results appeared even after active elimination of NOx contamination from laboratory ware. For example, plasma NOx concentrations did not increase during vasodilation mediated by the NO/cGMP pathway or after organ perfusion. A possible shift of NOx from plasma to erythrocytes (RBCs) as a cause of these phenomena has been excluded, leaving the destination of NOx (after leaving plasma) unknown. Kinetic analyses have revealed that steady state NOx concentrations in plasma and whole blood did not correlate with the NOx formation rate, but rather with the NOx elimination rate. Therefore, the supposition that the NO status is directly reflected by plasma NOx concentrations appears untenable. As nitrosothiols (R-SNOs), possible carriers of NO bioactivity, have been flagged as alternative indices of NO status in vivo, efforts have been made to detect these substances. When interference by ultrafiltration was eliminated, low molecular weight R-SNOs such as nitrosocystein and nitrosogluthathione were undetectable. However, a high-molecular weight R-SNO, nitrosoalbumin, was detected in human blood. Further research is required into the significance and practical use of nitrosoalbumin as a marker of NO in vivo.

Biomarkers↗
PubMed2003

Effects of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and chlorpromazine on NO3- transport via anion exchanger in erythrocytes: inertness of DIDS in whole blood.

We examined the effects of chlorpromazine on NO(3)(-) transport between erythrocytes (RBCs) and extracellular fluid. Chlorpromazine (10 microg/ml) did not influence NO(3)(-) movement in both whole blood and RBC suspension. Though an anion exchanger (AE1) inhibitor DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 100 microM) did not alter NO(3)(-) movement in whole blood, it inhibited the movement in a concentration-dependent manner in the RBC suspension. The inhibition was abrogated by plasma and albumin concentration-dependently. Our results indicated that chlorpromazine had no effect on NO(3)(-) transport through AE1 and that the inertness of DIDS on AE1 in whole blood is due to interference by albumin in plasma.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2003

A practical procedure for achieving a steady state of NOx concentration in plasma: with special reference to the NOx content of Japanese daily food.

To establish a concrete procedure to achieve a steady state plasma NOx concentration with Japanese daily food, NOx contents of about 200 types of food and beverages consumed daily were measured and NOx concentration in plasma was monitored till steady state after various degree of intake of NOx restricted food. The NOx content was found to be high in dark green leaved vegetables and low in grains, processed food, fresh and processed seafood. Tap water and mineral water were found to contain various amounts of NOx that were drastically reduced by treatment with a reverse osmosis column and remained in trace amounts after ion exchange column treatment. NOx content was low in drinks such as cola, but was extremely high in vegetable juice containing dark green leaved vegetables. The intake of high NOx drinks resulted in elevated plasma NOx concentration, but intake of low NOx drinks did not change the plasma NOx concentration. Based on these findings, a steady state could be achieved by 18 hours fasting after the intake of a moderately NOx-restricted diet (about 370, micromoles/day) and by 13 hours fasting after the intake of an extremely NOx-restricted diet (< 100 micromoles/day). NOx concentrations in randomly collected blood samples without these conditions were sometimes ten times higher than that at steady state. This procedure can be undertaken under normal Japanese daily life and is expected to be applicable even to outpatients.

Adult↗
PubMed2003

Association between coronary endothelial dysfunction and local inflammation of atherosclerotic coronary arteries.

We have examined a possibility whether or not severity and extent of coronary atherosclerosis may associate with degree of local inflammation in relation to endothelial dysfunction as is indicated by reduced NO formation. Blood samples were obtained from aortic root (Ao) and coronary sinus (CS) of 39 patients who underwent coronary angiography. Plasma NOx levels (nitrite + nitrate, stable NO end-products) were evaluated by HPLC-Griess system, and markers of inflammation, C-reactive protein (CRP) and serum amyloid A protein (SAA), were measured by Latex Turbidimetric Immunoassay. To evaluate the changes of these substances through coronary circulation, the percentage changes of respective markers [(CS - Ao) x 100/Ao] were calculated. The extent and severity of atherosclerosis of left coronary arteries were evaluated with Gensini Score (GS). The GS correlated with the percentage changes of NOx (r = -0.35, p < 0.05) and that of SAA (r = 0.43, p < 0.05) across coronary circulation, but not with changes in CRP. Moreover, the percentage changes of NOx correlated with that of SAA (r = -0.36, p < 0.05). These results indicated that severity and extent of coronary atherosclerosis related to degree of local inflammation which has a possible association with coronary endothelial dysfunction.

Biomarkers↗
PubMed2002

Paradoxical decrease in plasma NOx by L-arginine load in diabetic and non-diabetic subjects.

L-arginine, a substrate of nitric oxide synthase, was infused (30 g/300 ml/30 min) to patients with or without type 2 diabetes to examine whether or not endothelial dysfunction expressed as attenuated depressor response to the substrate in diabetic patients may accompany attenuated plasma NOx (NO2- and NO3-; an index of NO formation) elevation. Decrease in blood pressure by L-arginine was significantly smaller in diabetic patients than that in non-diabetic patients, and increase in plasma cGMP level in diabetic patients tended to be smaller and retarded than non-diabetic patients. However, plasma NOx decreased in both groups in a similar degree without changes in urinary NOx excretion, implying that NOx in plasma moved to other compartments. These results indicate that plasma NOx could not be solely used as an index of NO formation by L-arginine load and that this paradoxical decrease in plasma NOx would require further examination extending to other NOx compartments.

Arginine↗