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Biomedical subjects

S Kume

Publications and source records attributed to S Kume.

At least 91 records · Page 5Linked to original sources
PubMed1992

Thrombin-induced calcium oscillation in human platelets and MEG-01, a megakaryoblastic leukemia cell line.

Digital imaging microscopy revealed that human platelets show periodic intracellular Ca++ elevation in response to 0.01 U/ml thrombin. MEG-01, a megakaryoblastic leukemia cell line, also responded with oscillatory intracellular Ca++ elevation (0.7-1 times/min) to thrombin (0.001-0.003U/ml). Ca++ transients appears to be fused with higher thrombin doses. With extracellular Ca++ concentrations of 0.1 mM or less, Ca++ oscillation could not be elicited, or even when present, it disappeared after a few spikings of [Ca++]i. Extracellular Ca++ concentrations of 0.3 mM or more were required to facilitate ongoing Ca++ oscillation, suggesting an important role of Ca++ influx for Ca++ oscillation.

Blood Platelets↗
PubMed1992

Anti-platelet action of isoliquiritigenin, an aldose reductase inhibitor in licorice.

The mechanism was studied by which isoliquiritigenin, a new aldose reductase inhibitor purified from licorice (Glycyrrhizae radix), inhibits platelet aggregation. This new agent significantly inhibited the phosphorylation of 40,000- and 20,000-dalton proteins, and inhibited the formation of 12 (S)-hydroxy-5,8,10-heptadecatrienoic acid, 12-hydroxyeicosatetraenoic acid and thromboxane B2. The inhibitory effect of isoliquiritigenin on platelet aggregation in vitro was comparable to that of aspirin. Our findings may indicate that isoliquiritigenin elicits an anti-platelet action by inhibiting not only cyclooxygenase but also lipoxygenase or peroxidase activity in platelets. Isoliquiritigenin also showed an anti-platelet action in vivo. Isoliquiritigenin appears to be the only aldose reductase inhibitor with a significant anti-platelet action. Since the hyperaggregability of platelets has been implicated in the pathogenesis of diabetic complications, isoliquiritigenin may offer a unique benefit as an aldose reductase inhibitor.

Aldehyde Reductase↗
PubMed1992

Evaluation of platelet calcium ion mobilization by the use of various divalent ions.

Divalent ion mobilization in human platelets was evaluated with Fura-2 fluorescence changes induced by Ca2+, Sr2+, Ba2+ and Mn2+. Extracellular Ca2+, Sr2+ and Ba2+ all entered thrombin-stimulated platelets. These divalent ions were also able to refill the intracellular Ca2+ storage sites which had been depleted of Ca2+ by ionomycin treatment, and were released from the storage sites upon thrombin stimulation. However, only the refilling of the storage sites with Ca2+ and Sr2+, but not with Ba2+, were capable of suppressing the opening state of Ca2+ channels assessed with Mn2+ influx. Efflux of intracellularly accumulated divalent ions was observed with Ca2+ and Sr2+ but not with Ba2+. These findings indicate that there are subtle differences in the Ca(2+)-binding domains of the various systems involved in Ca2+ mobilization in platelets, some of which discriminate Ba2+ while accepting Sr2+.

Barium↗
PubMed1992

[Evaluation of platelet intracellular calcium ion concentrations with flow cytometry].

Platelet intracellular Ca++ ([Ca++] i) was measured by flow cytometry, using a new Ca(++)-sensitive fluorescent dye, fluo 3. The acetoxymethyl derivative of fluo 3, fluo 3 AM, was incorporated into platelets most efficiently in the presence of 1.5 micrograms/ml pluronic F-127, a surfactant often used to facilitate intracellular incorporation of lipophilic agents. [Ca++] i measurement with flow cytometry proved to be more sensitive than that with ordinary fluorescence spectrophotometers, detecting cells with elevated [Ca++] i at much lower agonist concentrations. Two-dimensional analysis using forward scatter intensities and [Ca++] i more clearly defined a subset of platelets responsive to low concentrations of agonists. In normal subjects [Ca++] i hardly changed in response to low-dose thrombin (0.002 U/ml), while it invariably showed a marked increase in response to high-dose thrombin (0.02 U/ml). Thus, these dose of thrombin were used to evaluate clinically whether platelets was hyper-reactive or hypo-reactive. Of 44 patients with diabetes mellitus, 6 patients had hypo-responsive platelets, while platelets were sensitive to low concentrations of thrombin in 3 patients. Since such differences cannot be detected by conventional spectrophotometric methods, [Ca++] i measurement by flow cytometry may prove useful tool for clinical evaluation of platelet function.

Blood Platelets↗
PubMed1991

Inhibition of platelet function by high-density lipoprotein from a patient with apolipoprotein E deficiency.

Apolipoprotein E-(apoE-) rich high-density lipoprotein (HDL) of normal subjects showed marked inhibitory effects on platelet aggregation and ATP release as compared with apoE-poor HDL, suggesting that apoE has inhibitory effects on platelet function (Desai et al. J. Lipid Res. 30:831, 1989; Higashihara et al. FEBS Lett. 282:82, 1991). A patient with apoE deficiency showed evidence of decreased platelet aggregability in platelet-rich plasma, but normal aggregability in washed platelets. Both patient's plasma and HDL fraction inhibited platelet aggregation of normal subjects. Patient's HDL reconstituted with recombinant apoE showed further inhibitory effects on platelet function. These results suggest that apoE is a potent, but not unique, inhibitory factor for HDL.

Adenosine Diphosphate↗
PubMed1991

Role of sodium ion gradient in facilitating extracellular calcium influx.

The effect of extracellular Na+ on Ca++ influx in activated human platelets was evaluated with two methods. One method employed computer-aided subtraction of the time course of intracellular Ca++ changes in the absence of extracellular Ca++ from that observed in the presence of extracellular Ca++. The other utilized intracellular Ca++ elevation following the addition of Ca++ to a platelet suspension already activated by thrombin in the absence of extracellular Ca++. Both methods demonstrated that depletion of extracellular Na+ significantly attenuated Ca++ influx in activated platelets. That extracellular Na+ modifies the opening state of Ca++ channels was further substantiated with an experiment with Sr++ instead of Ca++. A flow cytometric study confirmed the validity of these experiments. Neither Na+/H+ exchange nor changes in the membrane potential appeared to be related to the effect of extracellular Na+ on Ca++ influx, and the mechanism by which extracellular Na+ permits Ca++ mobilization remains to be elucidated.

Amiloride↗
PubMed1991

Two-step mobilization of arachidonic acid in platelet activation induced by low concentrations of TP 82, a monoclonal antibody against CD9 antigen.

Arachidonic acid mobilization in platelets activated by low concentrations (less than or equal to 1.6 micrograms/ml) of TP 82, a monoclonal antibody against CD9, appears to consist of two distinct phases. In the first phase, limited arachidonic acid release occurs concomitantly with a shape change induced by TP 82. This appears to be dependent upon phospholipase A2 activation, since it is well preserved in the presence of aspirin, which completely blocked both intracellular Ca2+ elevation and phosphatidic acid formation which would indicate phospholipase C activation. The Na+ Exchange was also found to participate in the first phase of arachidonic acid mobilization, since extracellular Na+ depletion and ethylisopropylamiloride, a specific inhibitor of the Na+/H+ exchanger, effectively blocked this limited mobilization of arachidonic acid. The second, much larger, phase of arachidonic acid mobilization occurs with the beginning of platelet aggregation. A limited amount of thromboxane A2 formed during the first phase of arachidonic acid release plays an important role in induction of the massive arachidonic mobilization in the second phase. Factors, as yet unidentified, also appear to work synergistically with thromboxane A2 to induce the full picture of arachidonic acid mobilization.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1991

The role of apoE in inhibitory effects of apoE-rich HDL on platelet function.

Apolipoprotein E- (ApoE-) rich high-density lipoprotein (HDL), which was prepared from the bound fraction of normolipemic volunteers on heparin-Sepharose and from a hyperalphalipoproteinemic patient, potently inhibited aggregation of human platelets in a dose-dependent fashion. Dimyristoyl phosphatidylcholine liposome with apoE (apoE.DMPC) also inhibited platelet aggregation, and incubation of washed platelets with apoE.DMPC resulted in the release of cholesterol into the supernatant in a time- and dose-dependent manner. These results suggest that apoE plays a major role in the inhibitory effect of apoE-rich HDL in platelet function, presumably due to the release of cholesterol from the plasma membrane.

Adenosine Triphosphate↗
PubMed1990

Effects of various inhibitors on platelet activation induced by TP 82, a CD 9 monoclonal antibody.

TP 82, a monoclonal antibody against CD 9 antigen, induced human platelet activation at concentrations higher than 0.4 microgram/mL in terms of aggregation, release of intracellular granule contents, production of arachidonic acid metabolites, and elevation of the intracellular Ca2+ concentration. The effects of a competitive inhibitor of ADP, acetylsalicylic acid, EGTA, and GRGDSP which blocks fibrinogen binding to IIb/IIIa complex suggested that each of released ADP, thromboxane A2, extracellular Ca2+, and close cell contact acts together to potentiate platelet activation induced by TP 82. While each of these inhibitors severely suppressed platelet activation induced by lower concentrations of the antibody (less than or equal to 0.8 microgram/mL), that induced by higher concentrations (greater than or equal to 3.2 micrograms/mL) was only partially blocked. Intracellular Ca2+ elevation was totally dependent upon the production of thromboxane A2, regardless of the antibody concentrations.

Adenosine Diphosphate↗
PubMed1990

Intracellular ionized calcium mobilization of CD 9 monoclonal antibody-activated human platelets.

Cytoplasmic Ca2+ mobilization in human platelets triggered by a CD 9 monoclonal antibody, TP82, was monitored by the Ca2(+)-sensitive photoprotein, aequorin and Ca2(+)-sensitive fluorophores, fura 2 and quin 2. Aequorin-indicated Ca2+ values were proportional to the concentration of TP82, which was in inverse proportion to the lag time before the onset of platelet aggregation and serotonin release. When fura 2 was used as a Ca2+ indicator, above a threshold concentration, the TP82-induced intracellular Ca2+ value remained unchanged even with increasing concentration. The findings obtained with quin 2 were compatible with the fact that the TP82-induced intracellular Ca2+ increase was largely dependent on the secondary effect of thromboxane A2. These findings may be clues to explain the marked difference in the Ca2(+)-response characteristics between the fluorescent indicators and aequorin as well as the properties of TP82-induced platelet activation.

Aequorin↗
PubMed1990

Modification of Na+/H+ exchanger in human platelets by 1,2-dioctanoylglycerol, a protein kinase C activator.

Protein kinase C activation in human platelets has a modulatory role in maintaining intracellular pH (pHi), by adjusting pHi at a particular value (7.22). Changes in pHi induced by protein kinase C appeared to be dependent upon the difference between H+ efflux catalyzed by the Na+/H+ exchanger and H+ production. The pHi recovery after acid loading was significantly facilitated by protein kinase C activation. Analysis of the rate constant for pHi recovery suggested that the turnover rate or the apparent affinity of the Na+/H+ exchanger for H+ was increased. Protein kinase C also decreased the Km value of the Na+/H+ exchanger for extracellular Na+. Thus, it is suggested that the role of protein kinase C in platelet pHi regulation is dual, adjusting the pHi value at a certain setpoint on the one hand, and increasing the rate constant of the Na+/H+ exchanger on the other.

Acids↗
PubMed1990

Mastoparan, a wasp venom, activates platelets via pertussis toxin-sensitive GTP-binding proteins.

Mastoparan induced limited release of serotonin from intact human platelets, while neither intracellular calcium ion elevation nor arachidonic acid mobilization was observed. Cytolysis induced by mastoparan was negligible in the concentration range that induced serotonin release. In digitonin-permeabilized cells, mastoparan induced Ca(++)-independent release of serotonin and Ca(++)-dependent arachidonic acid release. Both serotonin release and arachidonic acid release were reduced by pertussis toxin, suggesting that platelet activation induced by mastoparan is mediated by GTP-binding proteins.

Arachidonic Acids↗
PubMed1990

Ionomycin, a Ca++ ionophore, increases platelet volume independently of the Na+/H+ exchanger.

A Ca++-ionophore, ionomycin, increased the volume of human platelets suspended in a Ca++-containing buffer. This change in cell volume was dependent upon ionomycin and extracellular Ca++ concentrations, suggesting that the volume change occurs when the intracellular Ca++ reaches a certain level (greater than uM as determined by aequorin method). The ionomycin-induced volume increase was suppressed by replacement of extracellular Na+ with membrane-impermeable N-methyl-D-glucamine or Cs+, but not with Li+, K+, or Rb+. Ethylisopropylamiloride, a potent inhibitor of the Na+/H+ exchanger, had only weak inhibitory effect, and the apparent Km for Na+ was approximately 350 mM, which is much larger than that of the Na+/H+ exchanger. It is suggested that certain mechanisms other than the Na+/H+ exchanger are responsible for ionomycin-induced volume increase.

Blood Platelets↗
PubMed1989

Correlation of intracellular and extracellular calcium ion concentrations with synergy between 1,2-dioctanoyl-sn-glycerol and ionomycin in platelet arachidonic acid mobilization.

The potentiation by 1,2-dioctanoyl-sn-glycerol (DiC8) of ionomycin-induced platelet production of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was investigated in correlation with extracellular Ca2+ concentrations and increases in [Ca2+]i, as detected with aequorin and fura-2. Extracellular Ca2+ concentrations greatly influenced the production of arachidonic acid metabolites induced by DiC8 and ionomycin, while that induced by ionomycin alone was minimally affected by variation of the extracellular Ca2+ concentration. In the synergy between ionomycin and 20 microM DiC8, the optimal concentrations of ionomycin shifted from high to low with increasing concentrations of extracellular Ca2+, suggesting that there might be a range of optimal [Ca2+]i for the production of the arachidonic acid metabolites. This hypothesis was confirmed by simultaneous measurements of [Ca2+]i increases, and the production of the arachidonic acid metabolites. With the aequorin method, the optimal concentrations of [Ca2+]i fell to between 10 microM and 20 microM, and with the fura-2 method, it fell to between 800 nM and 1800 nM. Direct measurements of [14C]arachidonic acid release suggested that the DiC8-potentiated production of arachidonic acid metabolites induced by ionomycin was attributable to increased arachidonic acid release. Since ionomycin and DiC8 induced relatively low levels of phosphatidic acid production, an indicator of phospholipase C activation, it was suggested that the increased arachidonic acid release was largely dependent upon phospholipase A2. Synergy between DiC8 and ionomycin was also observed with aggregation and serotonin release. Aggregation was induced by lower concentrations of ionomycin, and appeared to be more dependent upon extracellular Ca2+, while serotonin release required higher concentrations of ionomycin, and variations in extracellular Ca2+ affected the response minimally. These findings suggest that the mechanisms underlying the synergy between protein kinase C activation and Ca2+ mobilization differ among the three functions evaluated in this study.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗