FACTORS AFFECTING DENSE AND ALPHA-GRANULE SECRETION FROM ELECTROPERMEABILIZED HUMAN PLATELETS - CA2+-INDEPENDENT ACTIONS OF PHORBOL ESTER AND GTP-GAMMA-S
FACTORS AFFECTING DENSE AND ALPHA-GRANULE SECRETION FROM ELECTROPERMEABILIZED HUMAN PLATELETS - CA2+-INDEPENDENT ACTIONS OF PHORBOL ESTER AND GTP-GAMMA-S
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DOI：
10.1091/mbc.1.13.1027
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发表时间：
1990-12-01
期刊：
影响因子：
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通讯作者：
HASLAM, RJ
中科院分区：
文献类型：
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作者：
COORSSEN, JR;DAVIDSON, MML;HASLAM, RJ
将含有5-羟基[C-14]色胺([C-14]5-HT)1的电通透性人血小板悬浮在含有三磷酸腺苷的谷氨酸介质中，并与钙缓冲液、佛波酯、鸟嘌呤核苷酸和凝血酶(不同组合)孵育10分钟。[C-14]5-羟色胺的释放和β-血栓球蛋白(β-TG)分别用于测量致密颗粒和α颗粒的分泌。Ca~(2+)单独刺激两种类型的颗粒分泌；当a-log[Ca~(2+)]无[(PCA)]为5.5时，半最大效应出现，当约80%的5-羟色胺和约50%的β-甘油三酯被释放时，在PCA为4.5时，分泌达到最大。加入PMA、鸟苷5‘-O-(3-硫代三磷酸)(GTP-γ-S)、GTP或凝血酶使5-羟色胺和β-甘油三酯分泌的钙量效曲线左移，并使最大分泌量略有增加。这些结果表明，与致密颗粒一样，α颗粒的分泌是一个受G蛋白、磷脂酶C和蛋白激酶C(PKC)顺序激活刺激的钙依赖过程。然而，高浓度的PMA和GTP-γ-S对Ca~(2+)的缺乏有明显的影响；100 nM的PMA大约释放20%的血小板5-羟色胺，但几乎没有β-TG，而100 mU-M的GTP-γ-S刺激各自大约25%的5-羟色胺的分泌。同时加入PMA可大大增强GTP-γ-S的上述效应。用[Gamma-P-32]ATP孵育的通透性血小板中pleckstrin的磷酸化作为PKC在分泌过程中活化的指标。在无钙条件下，100 nM PMA可引起Pleckstrin的最大磷酸化，而100mU-MGTP-γ-S的作用约为PMA的50%，GTP-γ-S和Ca~(2+)均不能增强100 nM PMA引起的Pleckstrin的磷酸化。这些结果表明，虽然激活PKC促进了分泌，但GTP-γ-S对密度和α颗粒的分泌都有额外的刺激作用，这不是由PKC介导的。对含有H-3[磷脂酰肌醇]的通透性血小板中H-3[肌醇磷酸]生成的测定表明，在无Ca~(2+)存在的情况下，GTP-γ-S不能刺激磷脂酶C。因此，在通透性的血小板中，GTP-γ-S除了通过这种磷脂酶外，还可以通过G-蛋白连接的效应因子来刺激PKC和促进分泌。
Electropermeabilized human platelets containing 5-hydroxy[C-14]tryptamine ([C-14]5-HT)1 were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [C-14]5-HT and beta-thromboglobulin (beta-TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log ]Ca2+ free[ (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta-TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta-TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca2+ - dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP-gamma-S had distinct effects in the absences of Ca2+ (pCa > 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta-TG, whereas 100-mu-M GTP-gamma-S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP-gamma-S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-P-32]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100-mu-M GTP-gamma-S was approximately 50% as effective as PMA; neither GTP-gamma-S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP-gamma-S exerted additional stimulatory effects on secretion from both denses and alpha-granules that were not mediated by PKC. Measurement of ]H-3[inositol phosphate formation in permeabilized platelets containing ]H-3[phosphoinositides showed that GTP-gamma-S did not stimulate phosphoinositide-specific phospholipase C in the absence of CA2+. It follows that in permeabilized platelets, GTP-gamma-S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.