哒嗪酮类药J-894对血小板激活时钙流动的影响

目的：探讨J－894对血小板功能的影响是否与钙有关。方法：用荧光钙离子指法剂观察J－894对血小板胞浆游离钙的影响。结果：J－894通过减少钙内流和释放明显抑制比凝血酶引起的血小板[Ca2 ]的升高,剂量与效应相关,J－894对钙内流的抑制比对钙释放的抑制作用强。结论：J－894抗血小板聚集可能主要是抑制钙内流。     

Effect of calmodulin antagonists on calcium and ethanol-induced sleeping time in mice

This investigation was carried out to determine if calcium prolongation of ethanol-induced sleep is mediated by calmodulin and a calmodulin-dependent protein kinase. The duration of ethanol-induced sleeping time in ddY male mice was measured following the administration of CaCl2 (20, 40, 80 and 200 mumol/kg, intraperitoneally (IP) both with and without the calmodulin antagonists, W-7: [N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide] (4.2 micrograms/mouse, intraventricular (IVT) or trifluoperazine (TFP; 1.8 micrograms/mouse, IVT). When CaCl2 was administered in a dose dependent manner the duration of ethanol-induced sleep was prolonged. The prolongation was antagonized by W-7 and TFP. When mice were treated with W-7 or TFP together with serotonin (5-HT; 15 nmol/mouse, IVT), dopamine (DA; 30 nmol/mouse, IVT) or norepinephrine (NE; 30 nmol/mouse, IVT), the sleeping time induced by ethanol and calcium was enhanced. This finding suggests that W-7 and TFP selectively inhibit the synthesis of 5-HT, DA and NE, but they do not affect other neuronal functions of these biogenic amines. The results would suggest a probable mechanism in which Ca++ prolongs ethanol-induced sleeping time by activating tyrosine hydroxylase and tryptophan hydroxylase through intracerebral calmodulin and calmodulin-dependent protein kinase, which subsequently raise the levels of 5-HT, DA and NE.     

Action of AD6 (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxycarbonylmethoxy coumarin) on human platelets in vitro

Summary The action of AD₆ was tested in vitro on human platelets by measuring beta-thromboglobulin (BTG), platelet factor 4 (PF₄) and thromboxane B₂ (TXB₂) release as well as aggregation. BTG and PF₄ release from blood anticoagulated with sodium citrate was inhibited by AD₆ during a 3 h incubation. Platelet rich plasma (PRP) was stimulated with ADP, collagen, sodium arachidonate, PAF, A23187 and epinephrine, while resuspended washed platelets (WP) were stimulated by thrombin. AD₆ (5–100 M) inhibited dose dependently aggregation, BTG, PF₄ and TXB₂ release induced by threshold concentration of all the tested aggregating agents; however AD₆ action could be overcome by increasing the concentration of the stimulating agents. After cyclo-oxygenase blockade by acetylsalicylic acid (ASA), PRP was stimulated by a supramaximal concentration of PAF. Under these circumstances we could observe a reversible aggregation and a partial release of BTG and PF₄, AD₆ was able to further reduce aggregation and release. Cyclic AMP accumulation induced in WP by prostacyclin was not modified by AD₆ (100 M), while theophylline greatly potentiated prostacyclin action. We conclude that AD₆ is an inhibitor of platelet activation in vitro. Its mode of action is different from cyclo-oxygenase blockade and provides inhibition of platelet activation by a number of different stimuli.     

CGS 9343B and W7 (calmodulin antagonists) inhibit KCl-induced increase in cytosolic free calcium and insulin secretion of RINm5F cells

Summary CGS 9343B:1,3-Dihydro-l-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin-4-yl)methyl)-4-piperidinyl]-2 H-benzimidazol-2-one maleate and W7: N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide) are calmodulin antagonists with different specificities. The effects of CGS 9343B and W7 on cytosolic free calcium concentration ([Ca²⁺]_i) and insulin release were investigated in rat insulinoma cells (RINm5F). As measured with the Quin-2 technique, preincubation with CGS 9343B (0.3–10 M) and W7 (5–50 M) concentration dependently decreased KCl (25 mM)-mediated accumulation of cytosolic calcium. Both, CGS 9343B (10 M) and W7 (50–100 M) almost abolished the alanine- and KCl-induced increase in [Ca²⁺]_i and significantly inhibited KCl (25 mM)- and alanine (10 mM)-mediated insulin release. W5 (100 LM), the chlorine-deficient analogue of W7 with decreased affinity for calmodulin, did not inhibit the KCl-induced increase in [Ca²⁺]_i and enhanced basal and KCl-mediated insulin release by 56% and 189%, respectively. Our data suggest that CGS 9343B and W7 inhibit the depolarization-induced calcium uptake and subsequent increase in [Ca²⁺]_i. Send offprint requests to H. P. T. Ammon at the above address     

血小板激活因子和银杏内酯B对洗涤兔血小板中cAMP含量的影响

研究了血小板激活因子(PAF)和PAF拮抗剂银杏内酯B对洗涤兔血小板中cAMP含量的作用. 结果表明PAF(0.1-1.0 μmol·L^-1)对血小板的基础cAMP水平无影响, 但对前列腺素E₁(PGE₁) 2 μmol·L^-1及4,5-二氢-6-［4-(1H-咪唑-1)苯基］-5-甲基-3-(2H)-哒嗪酮(CI-930) 20 μmol·L^-1引起的cAMP升高有显著的抑制作用. 银杏内酯B能完全拮抗PAF抑制PGE₁和CI-930升高cAMP的作用, IC₅₀分别为4.7和12.5 μmol·L^-1. 合用磷酸肌酸/磷酸肌酸激酶和阿司匹林对PAF和银杏内酯B的作用均无影响. 提示PAF对磷酸二酯酶的激活作用及腺苷酸环化酶的抑制作用是PAF的直接作用，与其同PAF受体结合有关.     

Effects of platelet activating factor and ginkgolid B on the cAMP levels in washed rabbit platelets

研究了血小板激活因子（ＰＡＦ）和ＰＡＦ拮抗剂银杏内酯Ｂ对洗涤兔血小板中ｃＡＭＰ含量的作用．结果表明ＰＡＦ（０．１－１．０μｍｏｌ·Ｌ－１）对血小板的基础ｃＡＭＰ水平无影响，但对前列腺素Ｅ１（ＰＧＥ１）２μｍｏｌ·Ｌ－１及４，５－二氢－６－［４－（１Ｈ－咪唑－１－）苯基］－５－甲基－３－（２Ｈ）－哒嗪酮（ＣＩ－９３０）２０μｍｏｌ·Ｌ－１引起的ｃＡＭＰ升高有显著的抑制作用．银杏内酯Ｂ能完全拮抗ＰＡＦ抑制ＰＧＥ１和ＣＩ－９３０升高ｃＡＭＰ的作用，ＩＣ５０分别为４．７和１２．５μｍｏｌ·Ｌ－１．合用磷酸肌酸／磷酸肌酸激酶和阿司匹林对ＰＡＦ和银杏内酯Ｂ的作用均无影响．提示ＰＡＦ对磷酸二酯酶的激活作用及腺苷酸环化酶的抑制作用是ＰＡＦ的直接作用，与其同ＰＡＦ受体结合有关．     

Exogenous GTP increases cyclic GMP and inhibits thrombin-induced aggregation of washed human platelets: comparison with ATP, adenosine and guanosine.

The effects of exogenous guanosine 5'-triphosphate (GTP), guanosine, adenosine 5'-triphosphate (ATP) and adenosine on platelet aggregation, serotonin secretion and cyclic nucleotide accumulation were studied using thrombin-stimulated washed human platelets. GTP (10 microM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion. The inhibition of aggregation was accompanied by an increase in platelet cyclic GMP. GTP did not affect cyclic AMP concentration. Adenosine (1 microM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion, and increased cyclic AMP. ATP at high concentrations (100 microM-1 mM) inhibited aggregation and serotonin secretion, and 1 mM ATP increased cyclic AMP. Guanosine was relatively ineffective in preventing aggregation and serotonin secretion and did not affect cyclic GMP. The rank order of inhibition of thrombin-induced aggregation of washed human platelets was adenosine > GTP > ATP > guanosine. In conclusion, exogenous GTP inhibits thrombin-induced aggregation and serotonin secretion of washed human platelets by increasing cyclic GMP. The results raise the possibility of a cell membrane site of action for GTP in platelets which mediates the activation of soluble guanylate cyclase suggesting that GTP may have a local antithrombotic effect also in vivo.     

Effect of cobaltous chloride on aggregation of platelets from normal and afibrinogenaemic human blood

The aggregation of normal human platelets in vitro induced by ADP was severely inhibited after preincubation of platelet-rich plasma (PRP) with CoCl2. Platelets from a patient with congenital afibrinogenaemia did not aggregate until fibrinogen was added. This recovered response was also inhibited by CoCl2. The impairment of aggregation seemed to be due to the action of cobalt on surrounding fibrinogen and not to a direct action on the platelets themselves. These results illustrate another aspect of the potential toxicity resulting from the use of cobaltous salts in treating the anaemia of renal failure, in which bleeding disorders have been reported.     

人血小板密度亚群在聚集反应,ATP释放及细胞内游离钙浓度的异质性(英文)

目的:研究人血小板密度亚群在聚集和释放反应及细胞内钙浓度的异质性。方法:Percoll间断梯度离心法。结果:正常人血小板分为高(HD),中(ID)和低(LD)密度三个亚群。血小板的大小随密度的降低而减小(r=0.978,P<0.01)。HD对凝血酶(Thr),ADP和血清素(5-HT)诱导的聚集反应明显强于LD(P<0.01),ATP释放反应,除5-HT无ATP释放外,与聚集反应的结果一致。尽管细胞内游离钙([Ca~(2 )]_i的静息水平各亚群相同,但Thr,ADP和5-HT的[Ca~(2 )]_i动员仍以HD最为明显。结论:人HD血小板的功能及活性均高于LD血小板。     

MK-447对家兔凝血酶诱导的血小板聚集,ATP释放及细胞内游离钙动员的影响(英文)

目的:研究MK-447对家兔凝血酶诱导的血小板聚集释放反应及单细胞内钙水平的影响.方法:利用浊度法及测定PRP中ATP的含量评价聚集和释放反应,以荧光图像法分析细胞内钙浓度.结果:MK-447仅使兔多血小板血浆(PRP)透光度降低(DLT),即血小板变形,单血小板[Ca~(2 )]_i轻度增加(160 nmol·L~(-1)),并不被依他酸3 mmol·L~(-1)抑制.MK-447消除凝血酶诱导的DLT,聚集和ATP释放增强,呈剂量依赖性,且凝血酶介导的[Ca2 ]_i由369 ±45 nmol·L~(-1)增加到621±121 nmol·L~(-1).结论:MK-447的血小板变形与其[Ca~(2 )]_i释放有关.MK-447增强凝血酶的血小板聚集和ATP释放.MK-447的这一作用可能于[Ca2 ]_i的协同作用有关.     

丹酚酸B镁盐对兔洗涤血小板聚集和5-HT释放反应的影响(英文)

AIM: To study the effects and mechanism of magnesium lithospermate B(MLB) on rabbit platelet aggregation and 5-HT release. METHODS: The platelet aggregation was determined by Born's method. Release of serotonin (5-HT) and formation of thromboxane A2 (TXA2) were measured by fluorophotometry and radioimmunoassay (RIA) respectively. Cytoplasmic free Ca2+ concentration ([Ca2+]i) in platelets was measured by Fura 2-AM fluorescence technique. RESULTS: In washed platelets, thrombin (200 U/L) or arachidonic acid (AA) (30 mumol/L)-induced aggregation was inhibited by MLB 50-800 mg/L in a concentration-dependent manner. In addition, MLB had more inhibitory effects on platelet aggregation in the absence of extracellular calcium with IC50 of 102 mg/L than in the presence of CaCl2 1 mmol/L with IC50 of 194 mg/L. MLB concentration-dependently decreased the thrombin-activated release of 5-HT, whereas it did not affect the formation of TXA2 in platelets. Furthermore, MLB not only inhibited the rise of [Ca2+]i in thrombin stimulated platelets, but decreased the [Ca2+]i in resting platelets. CONCLUSION: MLB inhibited the aggregation and 5-HT release in rabbit platelets and it is probably by attenuating intracellular calcium concentration.     

槲皮素对血小板聚集和胞浆游离钙的影响(英文)

目的:研究槲皮素对凝血酶诱导的血小板聚集和胞浆游离钙浓度的影响及钙对槲皮素的血小板聚集抑制效应的作用。方法:用荧光钙离子指示剂观察槲皮素对血小板胞浆游离钙的影响.结果:槲皮素明显抑制凝血酶诱导的血小板聚集和游离钙的升高.IC_(50)和95％可信区间分别为146.2(92.4～231.3)和78.5(49.5—124.4)μmol·L~(-1).槲皮素对血小板的抑制作用可被钙翻转.槲皮素对凝血酶诱导的钙释放无影响.结论:抑制钙内流是槲皮素抑制血小板聚集和[Ca~(2 )]_i升高的机制.     

Effects of calcium channel blockers and a calmodulin antagonist on contractions of guinea pig airways

Both antigen- and calcium ionophore A23187-induced airways contractions are dependent on increased concentrations of free intracellular calcium. Antigen, but not A23187, contracted airways in the absence of extracellular calcium, suggesting that antigen-induced airways contraction was partly dependent on the mobilization of intracellular calcium. A series of calcium entry blockers and a calmodulin antagonist were examined on airways contraction induced by antigen, A23187, histamine, and LTD4. Verapamil (10(-5)-3 X 10(-4) M) and nitrendipine (3 X 10(-5)-2.8 X 10(-4) M) dose-dependently inhibited antigen-induced contraction of trachea but only partly inhibited antigen-induced contraction of parenchyma and A23187-induced contractions of both trachea and parenchyma. Lanthanum chloride (up to 10(-4) M) and flunarizine (up to 2.1 X 10(-5) M) were relative ineffective. High concentrations of trifluoperazine (1-3 X 10(-4) M) were necessary to inhibit antigen- and A23187-induced airways contractions. Only verapamil and trifluoperazine were effective inhibitors of the responses to histamine and LTD4. In light of the relative ineffectiveness of the above calcium antagonists on the LTD4 dose-response curves, it is suggested that airways contraction is dependent on mobilization of intracellular calcium and that the action of nitrendipine and verapamil as inhibitors of antigen- and A23187-induced contractions is at the point of blocking mediator (i.e. LTC4 and LTD4) release. Verapamil and trifluoperazine also appear to affect contractions. However, the high concentrations of calcium channel and calmodulin antagonists to inhibit antigen- and A23187-induced contractions may limit their usefulness as anti-allergic, anti-asthmatic agents.     

Effect of calcium and calmodulin antagonists on contractile responses of the human uterine artery.

1. The dependence on extracellular calcium of contractile responses of intramyometrial arteries (0.5-2 mm diameter), as well as the effects of various types of calcium antagonists on these responses, were studied. Contractions were induced by K-depolarization (K) and noradrenaline (NA). 2. Whereas the K response was completely abolished in a calcium-free medium containing 2 mM LaCl3, the NA response was substantially maintained. 3. Nimodipine strongly inhibited the K response but had a relatively weak effect on the NA response; the IC50 values for the K and NA responses being 2 nM and 6 microM, respectively. Corresponding values for verapamil were about 0.7 and 10 microM. 4. Calmodulin antagonists, particularly trifluoperazine and flunarizine, caused a greater inhibition of the NA than of the K response. 5. These results indicate that besides the extracellular calcium which appears to be the major source of activator calcium, there is an intracellular pool of calcium which can be utilized to activate, albeit to a limited extent, drug-induced contractile responses.     

Effects of 5-HT released from platelets on thrombin-induced aggregation and ATP release in rabbit pla

Ｅｆｅｃｔｓｏｆ５ＨＴｒｅｌｅａｓｅｄｆｒｏｍｐｌａｔｅｌｅｔｓｏｎｔｈｒｏｍｂｉｎｉｎｄｕｃｅｄａｇｇｒｅｇａｔｉｏｎａｎｄＡＴＰｒｅｌｅａｓｅｉｎｒａｂｂｉｔｐｌａｔｅｌｅｔｓｉｎｖｉｔｒｏＬＩＢａｉＹａｎ１，ＬＩＷｅｎＨａｎ（Ｄｅｐａｒ...     

金雀异黄素对猪血小板聚集和胞浆游离钙的影响

AIM: To study the effects of genistein on aggregation and cytosolic free calcium concentration in platelets. METHODS: Using turbidimetry to analyse aggregation and using Fura-2 fluorescence technique to determine Ca2+ level. RESULTS: Genistein strongly inhibited the pig platelet aggregation induced by thrombin (250 U.L-1). When genistein concentrations were 5 and 20 mumol.L-1, the inhibition rates on the aggregation were 52% and 73%, respectively. Genistein inhibited the rise of cytosolic free calcium concentration in platelets stimulated by thrombin (500 U.L-1) in the presence of extracellular Ca2+ 1 mmol.L-1. When genistein concentrations were 10, 20, 40, and 80 mumol.L-1, the inhibition rates were 24%, 40%, 63%, and 65%, respectively, but no effect on thrombin-induced internal Ca2+ release from dense tubular system. CONCLUSION: Genistein is a potential anti-platelet agent, mainly due to an inhibition of Ca2+ influx.     

兔血小板释放的内源性5—HT对凝血酶诱导的血小板聚集和ATP释放的？…

AIM: To study the effects of arachidonic acid (AA)-induced endogenous serotonin (5-HT) release on platelet aggregation and ATP release by thrombin (Thr). METHODS: Platelet aggregation and release reaction were quantified by light transmission in platelet-rich-plasma (PRP) and the amount of ATP in medium. The effects of endogenous 5-HT were evaluated by the filtration of content in cuvette A (content A) containing endogenous 5-HT into cuvette B in which Thr-induced aggregation was observed in the absence/presence of ?(+/-)-5 (Z)-7-[3-endophenylsulfonylamino [2.2.1] bicyclohept-2-exo-yl]heptanoic acid, sodium salt? (S-145) or/and methysergide (Met). RESULTS: (1) AA 100 and 200 mumol.L-1 induced aggregation and ATP release in cuvette A. When the aggregation reached a peak, the content A directly caused platelet aggregation in cuvette B, and it was inhibited by S-145 100 nmol.L-1, Met 30 mumol.L-1, and inhibited more potently by S-145 + Met. (2) In the presence of S-145 100 nmol.L-1 in cuvette B, aggregations by Thr 0.1 and 0.3 IU.L-1 were enhanced (P < 0.01) by the filtrate, while Thr 0.5 IU.L-1-caused ATP release was suppressed (P < 0.01) without the effect on aggregation. Preincubation with S-145 and Met, the effects of the filtrate on aggregation and ATP release were abolished. (3) By prolongation of the time intervals between filtration and addition of Thr, the aggregation was enhanced and ATP release was reduced. CONCLUSION: Endogenous 5-HT was released from activated platelet and plays, in turn, a role in the regulation of platelet aggregation by the superimposition of cytosolic-free calcium ([Ca2+]i) and the feedback loop to regulate release reaction and calcium.     

Effects of vinblastine on malondialdehyde formation, serotonin release and aggregation in human platelets

Effects of the microtubular agent vinblastine on human platelet malondialdehyde formation, [14C]serotonin release and aggregation were studied in suspensions of [14C]serotonin-labelled platelets. Vinblastine caused dose-dependent inhibition of malondialdehyde formation and aggregation in platelet suspensions stimulated with thrombin, ADP or palmitaldehyde acetal phosphatidic acid (PGAP). Malondialdehyde formation, aggregation and [14C]serotonin release caused by threshold doses of thrombin were reduced but not abolished by 100 muM vinblastine; 30-100 muM vinblastine abolished ADP- and PGAP-induced malondialdehyde formation and [14C]serotonin released and transformed ADP- and PGAP-induced irreversible aggregation to a diminished reversible response. Arachidonate conversion to malondialdehyde catalysed by human platelet microsomes was inhibited by vinblastine and the cyclooxygenase inhibitors indomethacin and aspirin, but not by salicylate. Vinblastine inhibited the microsome-catalysed formation of malondialdehyde from prostaglandin H2. It is concluded that vinblastine inhibits the thromboxane pathway of arachidonate metabolism in stimulated platelets, consequently inhibiting release and aggregation, and that this effect of vinblastine may be, at least in part, independent of its antimicrotubular actions.     

Action of calcium antagonists and agonists on isolated human thoracic arteries used for coronary artery bypass grafting

BackgroundThe goal of this study was to investigate the modulation of the contraction-relaxation effects in isolated human thoracic artery samples of three calcium-channel antagonists, amlodipine (CAS [88150-42-9]), cerebrocrast (CAS [118790-71-9]) and diltiazem (CAS [42399-41-7]), and two calcium-channel agonists, CGP 28392 (CAS [89289-93-0]) and benzimidazole derivative. To estimate the endothelial function of the artery samples, carbachol, an agonist of muscarinic receptors, was used.MethodsThe experiments were conducted on isolated human thoracic artery samples, and their isometric contractions were recorded using an i-FOT10 force transducer. Cumulative concentration-contraction curves of the tested agents (10^?7 to 10^?4 M) were established.ResultsCarbachol at concentrations of 10^?7 to 10^?6 M did not cause any relaxation of artery rings precontracted by 10^?4 M phenylephrine, and at concentrations of 10^?5 and 10^?4 M, isometric contractions increased by 7% and 20%, respectively. In response to amlodipine, cerebrocrast and CGP28392, the contraction of artery samples increased significantly, whereas diltiazem and benzimidazole derivative caused their relaxation by ~55%.ConclusionThe obtained data indicate that the endothelium of the thoracic artery, which is used for coronary artery bypass grafting, is damaged by and may have some influence on the inadequate response of 1,4-dihydropyrine type calcium-channel antagonists.     

碘缺乏和甲状腺功能减退对大鼠仔鼠海马钙调蛋白表达的影响

目的观察碘缺乏和甲状腺功能减退对大鼠仔鼠海马钙调蛋白(calmodulin)表达的影响。方法健康2月龄雌性Wistar大鼠,交配妊娠后,取孕鼠28只,按体重随机分成对照组、碘缺乏组、甲状腺功能减退1和2组。自怀孕第6天(gestational day6,GD6)起,碘缺乏组饲以缺碘地区粮食配制的饲料,饮用自来水;甲状腺功能减退1和2组分别给予5和15ppm丙基硫尿嘧啶(propylthiouracil,PTU)饮水,饲以普通饲料,至生后第28天(postnatal day28,PN28)。分别于PN7、PN14和PN21时,每组随机取5只仔鼠,灌流固定大脑,进行组织病理切片和免疫组化染色,观察分析海马钙调蛋白表达。结果PN14和PN21时,在CA1和CA3区,15ppm组和碘缺乏组仔鼠海马钙调蛋白表达显著低于对照组(P0.05)。在DG区,钙调蛋白与对照组相比,差异无统计学意义。PN7时,各区均几乎观察不到阳性反应产物,各组间蛋白表达差异无统计学意义。结论在本试验条件下,碘缺乏和甲状腺功能减退可下调海马CA1和CA3区钙调蛋白蛋白表达。